ES2707288T3 - Particles of lentiviral vectors that have improved transduction efficiency for cells expressing DC-SIGN - Google Patents

Abstract

A method of generating a pseudotyped lentiviral vector particle comprising: (a) culturing in a culture medium comprising kifunensin, a virus packaging cell comprising: (1) a lentiviral vector genome comprising a polynucleotide encoding a sequence of expressible interest, (2) a polynucleotide encoding an alphavirus envelope glycoprotein that preferentially binds to dendritic cells expressing DC-SIGN and (3) a polynucleotide encoding a Vpx protein or a Vpr protein that retains activity SAMHD1 inhibitor; and (b) isolating a pseudotyped lentiviral vector particle that preferentially binds to dendritic cells expressing DC-SIGN, wherein said lentiviral vector particles are more highly mannosylated compared to a control composition of the same pseudotyped lentiviral vector particles. prepared in the absence of a mannosidase inhibitor.

Description

DESCRIPTION

Particles of lentiviral vectors that have improved transduction efficiency for cells expressing DC-SIGN

Field of the invention

The present disclosure relates to materials and methods useful for generating improved pseudotyped lentiviral vector particles.

Background

Dendritic cells (DC) are essential antigen presenting cells for the initiation and control of immune responses. DC can capture and process antigens, migrate from the periphery to a lymphoid organ and present the antigens to resting T cells in a restricted form of the major histocompatibility complex (MHC). These cells derive from the bone marrow (BM) and have dendritic morphology and high mobility. The discovery of DC as specialized antigen presenting cells (APCs) has prompted attempts at CD-based immunization / vaccination strategies that involve directing DC to present specific antigens. Vectors based on recombinant viruses have been developed as a mechanism to directly deliver a gene encoding an antigen / antigens designated to host cells. By inducing a desired adaptive immune response, the gene product expressed provides a therapeutic benefit.

The challenges to achieving a safe and effective system include designing a vector that efficiently targets a desired set of host cells, providing an adequate delivery system and expressing a desired antigen that produces an effective immune response so that it can be widely used in a whole population of designated human subjects.

The envelope glycoproteins of the Sindbis virus and other alphaviruses described herein are incorporated into the lipid bilayer of the viral particle membrane. Typically, the viral membrane (envelope) includes multiple copies of trimers of two glycoprotein heterodimers, E1 and E2, which are produced by cleavage of a single precursor protein. The precursor protein comprises, from its N to C end, the E3, E2, 6K and E1 proteins. The small E3 glycoprotein serves as a signal sequence for the translocation of the E2 protein in the membrane, and is cleaved from E2 by furin or some other serine proteinase dependent on Ca2 +. The 6K protein serves as a signal sequence for the translocation of the E1 protein in the membrane and is then cleaved from the precursor protein. WO 2008/011636 and US 2011/0064763 describe lentiviral packaging systems.

Brief summary of the invention

The inventors have discovered that lentiviral vector particles having two characteristics (a) pseudotyped with a highly-mannosylated alphavirus glycoprotein and (b) comprising a Vpx protein, have an unexpectedly improved transduction efficiency for expressing cells DC-SIGN. These particles infect cells expressing DC-SIGN, in particular dendritic cells, significantly more efficiently than particles of lentiviral vectors having only one of these two characteristics.

The present invention provides a method for generating a pseudotyped lentiviral vector particle comprising: (a) culturing, in a culture medium comprising kifunensine, a virus packaging cell comprising: (1) a lentiviral vector genome comprising a polynucleotide encoding an expressible sequence of interest, (2) a polynucleotide encoding an alphavirus envelope glycoprotein that binds preferentially to dendritic cells expressing DC-SIGN, and (3) a polynucleotide that encodes a Vpx protein or a Vpr protein that retains the inhibitory activity of SAMHD1; and (b) isolating a pseudotyped lentiviral vector particle that preferentially binds dendritic cells expressing DC-SIGN, wherein said lentiviral vector particles are more highly mannosylated compared to a control composition of the same pseudotyped lentiviral vector particles. prepared in the absence of a mannosidase inhibitor. The present disclosure also sets forth ("provides") a series of compositions and methods as described hereinafter. However, the invention is defined in the appended claims.

In particular cases, highly mannosylated pseudotyped lentiviral vector particles comprising a Vpx protein and a lentiviral genome comprising a sequence of interest (e.g., a polynucleotide encoding an antigen) are provided.

Methods for generating particles of pseudotyped lentiviral vectors.

One aspect of the disclosure provides a method for generating a pseudotyped lentiviral vector particle comprising: (a) culturing in a kifunensin culture medium a virus packaging cell comprising: (1) a lentiviral vector genome comprising a polynucleotide that encodes an exogenous antigen, (2) a polynucleotide encoding an alphavirus glycoprotein that preferentially binds DC-SIGn expressing cells, and (3) a polynucleotide encoding a SAMHD1 inhibitor; and (b) isolating a pseudotyped lentiviral vector particle that preferentially binds to cells expressing DC-SIGN.

Another aspect of the disclosure provides a method for generating a pseudotyped lentiviral vector particle comprising: (a) culturing in a culture medium comprising kifunensine a virus packaging cell comprising: (1) a lentiviral vector genome comprising a polynucleotide encoding an exogenous antigen, (2) a polynucleotide encoding a Sindbis E2 glycoprotein that preferentially binds to dendritic cells expressing DC-SIGN, and (3) a polynucleotide that encodes a Vpx protein or a Vpr protein that retains the inhibitory activity of SAMHD1; and (b) isolating a pseudotyped lentiviral vector particle that preferentially binds dendritic cells expressing DC-SIGN. In some embodiments, glycoprotein E2 is 90% identical to SEQ ID NO: 30 [SIN-Var1]. In some embodiments, (i) residue 160 of glycoprotein E2 is absent or is an amino acid other than glutamic acid, (ii) one or more of residues 70, 76 or 159 of the glycoprotein variant E2 is a non-basic residue , and (iii) the E2 glycoprotein variant is not part of a fusion protein with the E3 glycoprotein of the Sindbis virus. In some embodiments, glycoprotein E2 is SEQ ID NO: 30 [SIN-Var1].

In some or any of the embodiments described herein, the Vpx protein comprises an amino acid sequence that is at least 80% identical to SIVmac Vpx (SEQ ID NO: 44).

In some or any of the embodiments described herein, the Vpx protein comprises at least 90% amino acid sequence identical to SIVmac Vpx (SEQ ID NO: 44), SIVsm Vpx (SEQ ID NO: 45), SIVrcm Vpx ( SEQ ID NO: 46), or HIV-2 Vpx (SEQ ID NO: 47).

In some or any of the embodiments described herein, the Vpr protein comprises at least 90% amino acid sequence identical to SIVdeb Vpr (SEQ ID NO: 48) or SIVmus Vpr (SeQ ID NO: 49).

In some or any of the embodiments described herein, the antigen is a tumor-specific antigen or a virus-specific antigen. In some or any of the embodiments described herein, the tumor specific antigen is selected from the group consisting of NY-ESO-1, MAGE, p. eg, MAGE-A3 and MAGE-A1, MART-1 / Melan-A, BAGE, RAGE, gp100, gp75, mda-7, tyrosinase, tyrosinase-related protein, p. eg, TRP2, renal cell carcinoma antigen, 5T4, SM22-alpha, carbonic anhydrase I, carbonic anhydrase IX (also known as G250), HIF-1alpha, HIF-2alpha, VEGF, prostate-specific membrane antigen ( PSMA), prostate specific antigen (PSA), prostatic acid phosphates, prostate epithelial antigen with six transmembrane domains (STEAP), NKX3.1, telomerase enzyme, survivin, mesothelin, mutated ras, bcr / abl rearrangement, Her2 / neu, mutated p53, wild type p53, cytochrome P4501B1, N-acetylglucosaminyltransferase-V, human papillomavirus E6 protein, human papillomavirus E7 protein, carcinoembryonic antigen, Merkel cell virus T antigen oncoproteins and alpha-fetoprotein. In some or any of the embodiments described herein, the virus-specific antigen is an HIV antigen, an SIV antigen, an adenovirus antigen, an enterovirus antigen, a coronavirus antigen, a calicivirus antigen, a distemper virus antigen, an Ebola virus antigen, a flavivirus antigen, a hepatitis virus antigen, a herpesvirus antigen, an infectious peritonitis virus antigen, an influenzavirus antigen, a virus antigen leukemia, a Marburg virus antigen, an orthomyxovirus antigen, a papillomavirus antigen, a parainfluenza virus antigen, a paramyxovirus antigen, a parvovirus antigen, a pestivirus antigen, a picornavirus antigen, a poliovirus antigen , a smallpox virus antigen, a polyomavirus antigen, a rabies virus antigen, a reovirus antigen, a retrovirus antigen or a rotavirus antigen.

In some or any of the embodiments described herein, the genome of the lentiviral vector further comprises a nucleotide sequence encoding a second antigen. In some or any of the embodiments described herein, the first and second antigens are expressed as a fusion protein comprising a peptide A2 of self-cleavage between the two antigens. In some or any of the embodiments described herein, the self-cleaving peptide A2 comprises the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 57. In some or any of the embodiments described herein, the first antigen is MAGE-A3 and the second antigen is NY-ESO-1.

In some or any of the embodiments described herein, kifunensine is present in the culture medium in a concentration of about 0.01 pg / ml to about 1 mg / ml. In some or any of the embodiments described herein, kifunensine is present in the culture medium in a concentration of about 0.1 pg / ml to about 1 pg / ml. In some or any of the embodiments described herein, kifunensine is present in the culture medium in a concentration of about 0.25 pg / ml to about 2 pg / ml.

In some or any of the embodiments described herein, the virus packaging cell further comprises: (i) a polynucleotide comprising the gag and pol genes; and (ii) a polynucleotide encoding a rev. In some or any of the embodiments described herein, the gag and pol genes are human optimized codons and comprise a non-optimized window around the position 1228 to 1509 of SEQ ID NO: 54. In some or any of the embodiments described herein, the polynucleotide comprising the gag and pol genes lacks a functional rev response element (RRE). In some or any of the embodiments described herein, the polynucleotide comprising the gag and pol genes lacks a functional RRE because the RRE has been deleted. In some or any of the embodiments described herein, the pol gene encodes an inactive integrase enzyme. In some or any of the embodiments described herein, the integrase enzyme has a D64V mutation. In some or any of the embodiments described herein, the polynucleotide encoding the Vpx protein or the Vpr protein is on the same or different plasmid as the polynucleotide encoding the rev protein, or the polynucleotide comprising the gag and pol genes. .

In some or any of the embodiments described herein, the genome of the lentiviral vector is obtained from HIV-1.

In some or any of the embodiments described herein, the genome of the lentiviral vector has an inactivated 3 'long terminal repeat (LTR) or a self-activating 3' long terminal repeat (LTR). In some or any of the embodiments described herein, the genome of the lentiviral vector comprises an U3 element that lacks at least one enhancer sequence, one TATA box, one Sp1 site, one NK-kappa B site or one stretch of polypurine (PPT).

In some or any of the embodiments described herein, the genome of the lentiviral vector comprises the nucleotide sequence of any one of SEQ ID NO: 21 [SIN vector], 22 [vector 703] or 23 [vector 704].

In some or any of the embodiments described herein, the genome of the lentiviral vector further comprises a nucleotide sequence that encodes a dendritic cell maturation / stimulation factor. In some or any of the embodiments described herein, the maturation / stimulation factor of the dendritic cells is selected from the group consisting of GM-CSF, IL-2, IL-4, IL-6, IL-7, IL-15, IL-21, IL-23, TNFa, B7.1, B7.2, 4-1BB, CD40 ligand and CD40 inducible by drug.

In some or any of the embodiments described herein, the nucleotide sequence encoding an antigen is operatively linked to a promoter selected from the group consisting of the human ubiquitin-C (UbiC) promoter, the immediate early promoter of cytomegalovirus. (CMV), the Rous sarcoma virus promoter (RSV) and the tetracycline sensitive promoter. In some or any of the embodiments described herein, the promoter is an introns deficient promoter. In some or any of the embodiments described herein, the introns deficient promoter is a UbiC

Related aspects of the description provide a lentiviral vector particle produced by any of the methods mentioned above.

Compositions comprising particles of pseudotyped lentiviral vectors.

Another aspect of the disclosure provides a composition comprising pseudotyped lentiviral vector particles comprising (a) an inhibitor of SAMHD1, (b) an exogenous polynucleotide encoding an antigen, and (c) a envelope glycoprotein that preferentially binds to cells dendritic cells expressing DC-SIGN, wherein at least 60%, or at least 70%, or at least 80%, preferably at least 90% of the N-linked glycans in said composition comprise a Man ⁵ structure. Mang, preferably Mang.

Another aspect of the disclosure provides a composition comprising pseudotyped lentiviral vector particles comprising (a) a Vpx protein, (b) an exogenous polynucleotide encoding an antigen, and (c) a envelope glycoprotein that preferentially binds to dendritic cells expressing DC-SIGN, wherein at least 80% of the glycans linked to N in said composition comprise a Mang structure.

In some or any of the embodiments described herein, the Vpx protein comprises an amino acid sequence that is at least 80% identical to the SIVmac Vpx protein (SEQ ID NO: 44).

In some or any of the embodiments described herein, the Vpx protein comprises at least 90% amino acid sequence identical to SIVmac Vpx (SEQ ID NO: 44), SIVsm Vpx (SEQ ID NO: 45), SIVrcm Vpx ( SEQ ID NO: 46), or HIV-2 Vpx (SEQ ID NO: 47).

In some or any of the embodiments described herein, the Vpr protein comprises at least 90% amino acid sequence identical to SIVdeb Vpr (SEQ ID NO: 48) or SIVmus Vpr (SeQ ID NO: 49). In some or any of the embodiments described herein, the pseudotyped lentiviral vector particle infects dendritic cells expressing DC-SIGN with an in vitro transduction efficiency of at least 1%, or at least 5%, or at least 10%. %, or at least 20%. See, p. eg, the procedure of example 8.

In some or any of the embodiments described herein, the glycoprotein is an E2 glycoprotein of the Sindbis virus. In some or any of the embodiments described herein, the glycoprotein E2 has at least 90% identity with SEQ ID NO: 30 [SIN-Var1]. In some or any of the embodiments described herein, (i) residue 160 of glycoprotein E2 is absent or is an amino acid other than glutamic acid, (ii) one or more of residues 70, 76 or 159 of the E2 glycoprotein variant is a non-basic residue, and (iii) the E2 glycoprotein variant is not part of a fusion protein with E3 glycoprotein Sindbis virus.

In some or any of the embodiments described herein, the antigen is a tumor-specific antigen or a virus-specific antigen. In some or any of the embodiments described herein, the tumor specific antigen is selected from the group consisting of NY-ESO-1, MAGE, p. eg, MAGE-A3 and MAGE-A1, MART-1 / Melan-A, BAGE, RAGE, gp100, gp75, mda-7, tyrosinase, tyrosinase-related protein, p. eg, TRP2, renal cell carcinoma antigen, 5T4, SM22-alpha, carbonic anhydrase I, carbonic anhydrase IX (also known as G250), HIF-1alpha, HIF-2alpha, VEGF, prostate-specific membrane antigen ( PSMA), prostate specific antigen (PSA), prostatic acid phosphates, prostate epithelial antigen with six transmembrane domains (STEAP), NKX3.1, telomerase enzyme, survivin, mesothelin, mutated ras, bcr / abl rearrangement, Her2 / neu, mutated p53, wild type p53, cytochrome P4501B1, N-acetylglucosaminyltransferase-V, human papillomavirus E6 protein, human papillomavirus E7 protein, carcinoembryonic antigen, Merkel cell virus T antigen oncoproteins and alpha-fetoprotein. In some or any of the embodiments described herein, the virus-specific antigen is an HIV antigen, an SIV antigen, an adenovirus antigen, an enterovirus antigen, a coronavirus antigen, a calicivirus antigen, a distemper virus antigen, an Ebola virus antigen, a flavivirus antigen, a hepatitis virus antigen, a herpesvirus antigen, an infectious peritonitis virus antigen, an influenzavirus antigen, a virus antigen leukemia, a Marburg virus antigen, an orthomyxovirus antigen, a papillomavirus antigen, a parainfluenza virus antigen, a paramyxovirus antigen, a parvovirus antigen, a pestivirus antigen, a picornavirus antigen, a poliovirus antigen , a smallpox virus antigen, a rabies virus antigen, a reovirus antigen, a retrovirus antigen or a rotavirus antigen.

In some or any of the embodiments described herein, the genome of the lentiviral vector further comprises a nucleotide sequence encoding a second antigen. In some or any of the embodiments described herein, the first and second antigens are expressed as a fusion protein comprising a peptide A2 of self-cleavage between the two antigens. In some or any of the embodiments described herein, the self-cleaving peptide A2 comprises the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 57. In some or any of the embodiments described herein, the first antigen is NY-ESO-1 and the second antigen is MAGE-A3.

In some or any of the embodiments described herein, the genome of the lentiviral vector is obtained from HIV-1.

In some or any of the embodiments described herein, the genome of the lentiviral vector has an inactivated 3 'long terminal repeat (LTR) or a self-activating 3' long terminal repeat (LTR). In some aspects, the genome of the lentiviral vector comprises an U3 element that lacks at least one enhancer sequence, a TATA box, an Sp1 site, an NK-kappa B site or a polyprin (PPT) stretch.

In some or any of the embodiments described herein, the genome of the lentiviral vector comprises the nucleotide sequence of any of SEQ ID NO: 21 [SIN vector], 22 [vector 703] or 23 [vector 704].

In some or any of the embodiments described herein, the genome of the lentiviral vector further comprises a nucleotide sequence that encodes a dendritic cell maturation / stimulation factor. In some or any of the embodiments described herein, the maturation / stimulation factor of the dendritic cells is selected from the group consisting of GM-CSF, IL-2, IL-4, IL-6, IL-7, IL-15, IL-21, IL-23, TNFa, B7.1, B7.2, 4-1BB, CD40 ligand and CD40 inducible by drug.

In some or any of the embodiments described herein, the nucleotide sequence encoding an antigen is operatively linked to a promoter selected from the group consisting of the human ubiquitin-C (UbiC) promoter, the cytomegalovirus immediate early promoter. (CMV), the Rous sarcoma virus (RSV) promoter and the tetracycline sensitive promoter. In some or any of the embodiments described herein, the promoter is an introns deficient promoter. In some or any of the embodiments described herein, the introns deficient promoter is a human ubiquitin-C promoter (UbiC).

In some or any of the embodiments described herein, the particles of pseudotyped lentiviral vectors have an IU of at least 105 / ml.

In some or any of the embodiments described herein, the composition further comprises an immunostimulatory agent.

In some or any of the embodiments described herein, the composition further comprises an adjuvant. For example, as described herein, adjuvants include alum or monophosphoryl lipid A (MPL) 3 De-O-acylated. The classes of adjuvants described herein include (a) aluminum salts, (b) oil-in-water emulsion formulations, optionally with or without other specific immunostimulatory agents, such as muramyl peptides or other components of the bacterial cell wall. , (c) saponin adjuvants, including ISCOM (immunostimulatory complexes) and ISCOMATRIX; (d) Freund's complete adjuvant (CFA) and incomplete Freund's adjuvant (IFA); (e) cytokines; and (f) adjuvants of formula (I).

wherein the residues A1 and A2 are independently selected from the group of hydrogen, phosphate and phosphate salts. Sodium and potassium are example counterions for the phosphate salts. The residues R1, R2, R3, R4, R5 and R6 are independently selected from the group of hydrocarbyl having from 3 to 23 carbons, represented by C ³ -C ²³ . For clarity, it will be explained that when a remainder is selected "independently of" a specific group that has several members, it should be understood that the member chosen for the first remainder has no impact or limits in any way the election of the selected member for the second rest. The carbon atoms to which R1, R3, R5 and R6 are attached are asymmetric and, therefore, may exist in the stereochemistry R or S. In one embodiment all those carbon atoms have the R stereochemistry, while in another embodiment embodiment all those carbon atoms have the stereochemistry S.

In various embodiments of the description, the adjuvant has the chemical structure of the formula (I) but the residues A1, A2, R1, R2, R3, R4, R5 and R6 are selected from subsets of the options previously provided with these residues, where these subsets are then identified by E1, E2, etc.

E1: A ¹ is phosphate or phosphate salt and A ² is hydrogen.

E2: R1, R3, R5 and R6 are D ²¹ alkyl; and R2 and R4 are C ⁵ -C ²³ hydrocarbyl.

E3: R1, R3, R5 and R6 are D ¹⁷ alkyl; and R2 and R4 are C ⁷ -C ¹⁹ hydrocarbyl.

R1, R3, R5 and R6 are D ¹⁵ alkyl; and R2 and R4 are hydrocarbyl C ⁹ -C ^17.

R1, R3, R5 and R6 are alkyl ¹³ ; and R2 and R4 are C ¹¹ -C ¹⁵ hydrocarbyl.

R1, R3, R5 and R6 are alkyl ¹⁵ ; and R2 and R4 are C ¹¹ -C ¹⁷ hydrocarbyl.

R1, R3, R5 and R6 are D ¹³ alkyl; and R2 and R4 are C ⁹ -C ¹⁵ hydrocarbyl.

R1, R3, R5 and R6 are alkyl ■ C ^20; and R2 and R4 are C ¹² -C ²⁰ hydrocarbyl.

R1, R3, R5 and R6 are alkyl and R2 and R4 are hydrocarbyl C ^13.

E10: R1, R3, R5 and R6 are undecyl and R2 and R4 are tridecyl.

In certain options, each of E2 to E10 is combined with the E1 embodiment, and / or the hydrocarbyl groups of E2 to E9 are alkyl groups, preferably straight chain alkyl groups. A preferred adjuvant is E1 in combination with E10, where (i) A ¹ is phosphate or phosphate salt and A ² is hydrogen and (ii) R 1, R 3, R 5 and R 6 are undecyl and R 2 and R 4 are tridecyl.

In some or any of the embodiments described herein, the envelope glycoproteins also bind to cells expressing mouse SIGNR1.

In some or any of the embodiments described herein, the pseudotyped lentiviral vector particles also more efficiently transduce cells expressing mouse SIGNR1 compared to cells that do not express mouse SIGNR1.

Other features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and the specific examples, while indicating specific embodiments of the description, are provided by way of illustration only, since various changes and modifications within the spirit and scope of the description will be apparent to the experts in the art. the technique from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A and 1B illustrate the ability of pseudotyped lentiviral vector particles produced in the presence of different mannosidase inhibitors (eg, kifunensine, DMNJ and swainsonin) to infect HT1080 cells stably expressing the DC-SIGN receptor (1A) or lack of DC-SIGN (1B). The efficacy of the infection was evaluated by determining the expression of GFP from the lentiviral vector. The y-axis is the percentage of cells positive for GFP.

Figures 2A and 2B illustrate the capacity of pseudotyped lentiviral vector particles produced in the presence of 400 pg / ml DMNJ or different concentrations of kifunensine, to infect HT1080 cells stably expressing the DC-SIGN receptor (2A) or lacking of DC-SlGN (2B). The efficacy of the infection was evaluated as in Figure 1.

Figure 3A is a diagram illustrating the substrate specificity for PNGaseF and EndoH. PNGasaF is a general endoglycosidase that will cleave all N-linked glycosylation, independently of the glycosylation profile. EndoH is a specialized endoglycosidase that will only cleave N-linked glycosylation with high mannose content. EndoH only cleaves N-linked glycosylation with high mannose content (ie, EndoH will target 2 out of 4 sites on SINVarl in the absence of kifunensine, and 4 out of 4 sites on SINVarl in the presence of kifunensine). Figure 3B illustrates the results of an experiment to determine the glycosylation status of the glycoproteins in particles of pseudotyped lentiviral vectors produced in the presence of kifunensine or DMNJ using gel shifts by performing on an SDS-PAGE gel and immunoblotting with antibody against the viral envelope of Sindbis.

Figure 4A illustrates the ability of pseudotyped lentiviral vector particles produced in the presence of 400 pg / ml DMNJ or different concentrations of kifunensine to infect HT1080 cells stably expressing the DC-SIGN receptor. The efficacy of the infection was evaluated as in Figure 1. Figure 4B illustrates the results of an experiment to determine the glycosylation status of glycoprotein E2 in particles of pseudotyped lentiviral vectors produced in Figure 4A.

Figure 5A is a western blot (anti-HA) of SlVmacVpx labeled with HA expressed in 293T cells. It was cloned into a mammalian expression vector driven by a CMV promoter (construct called pENV-SlVmacVpx). Figure 5B is a Western blot (anti-HA) of viral protein extracts from viral particles comprising SlVmacVpx labeled with HA. 100 ng of p24 was loaded per well into a gel for immunoblotting with anti-HA antibody. Anti-p24 antibody was used as a loading control.

Figure 6 is a scatter diagram of transduction events measured by selection in dendritic cells that were positive for CD11c and evaluation of the percentage of cells positive for GFP (x axis, as a result of infection by defective integration lentivirus, pseudotyped VSV- G with or without Vpx) with DC-SIGN on the y axis. Figure 7 is a scatter plot of transduction events measured by selection in dendritic cells that were positive for CD11c and evaluation of the percentage of cells positive for GFP (x-axis) as a result of infection by the competent integration lentivirus, pseudotyped VSV -G with or without Vpx) with DC-SIGN on the y-axis.

Figure 8 is a scatter diagram of transduction events measured by selection in dendritic cells infected with defective integration lentiviruses, pseudotyped SINvarl with or without Vpx and produced in the presence of absence of kifunensine. The y-axis measures cells that were positive for CD11c or DC-SIGN and the x-axis measures the percentage of cells positive for GFP.

Figure 9A is a Western blot of Sindbis virus glycoproteins prepared according to Example 1 without treatment (lanes 1-3), DMNJ 400 pg / ml (lanes 4-6), or 1 pg / ml kifunensine (lanes 7). -9). The particles were not treated with endoglycosidase (lanes 1, 4, 7), incubated with EndoH for 1 hour (lanes 2, 5, 8) or incubated with PNGasaF for 1 hour (lanes 3, 6, 9). The samples were then analyzed using a gel shift assay and immunoblotting with antibody against the Sindbis virus envelope. Figure 9B (without mannosidase inhibitor) is an intensity profile of each band of bands 1-3 and their location in the band. The Figure 9C (treated with DMNJ) is an intensity profile of each band of bands 4-6 and its location in the band. Figure 9D (treated with kifunensine) is an intensity profile of each band of bands 7-9 and its location in the band.

For Figure 10A, HuC-SIGN 293T cells were transduced with vectors packaged with wild-type (WT) or defective integrase (D64V) and a vector genome containing extended 3'PPT (703) or 3'PPT deletion (704). ). At 48 hours after transduction, an analysis of Alu-PCR nested in the genomic DNA extracted from the transduced cells was carried out. The error bars indicate the standard error of the average of the transductions made in triplicate. Figures 10B and 10C demonstrate the integration rate of WT / 703 and D64V / 704 vectors encoding GFP-T2A-NeoR using two independent methods. For Figure 10B, HT1080 huDC-SIGN cells were transduced with serial dilutions of the indicated vectors. The transduced cells were cultured under selection with G418 and the neomycin-resistant colonies were counted, representing individual integration events. The integration events were calculated as described in Example 10. For Figure 10C, HT1080 huDC-SIGN cells were transduced with the indicated vectors. Flow cytometry was performed at multiple measurement times after transduction to determine the percentage of cells expressing GFP. The error bars indicate the standard error of the mean of the flow cytometry in triplicate.

Figure 11: Human PBMCs were treated with GM-CSF and IL-4 for 3 days, then incubated with 20 ng of p24 of ID-VP02 encoding GFP. At the time of vector addition, the culture consisted mainly of DC, B cells and T cells. Three days after transduction, the surface markers were analyzed in the cells and classified as DC (CD11cpos) 6%, cells B (CD11cneg, CD19pos) 10% or T cells (CD11cneg, cD3 £ pos) 80%. Transduction events were measured by evaluating the percentage of GFP-positive cells within each cell population. The numbers in the graphs are the percentage of cells within the GFP + selection.

Figure 12: Represents a part of the vector genome (top) that contains long terminal repeats (LTR) at each end. The splice donor (SD) and splice acceptor (SA) sites flank the psi packaging signal (V), the partial gag sequence and the Rev response element (RRE). The components between the antigen promoter (promoter) and the 3 'LTR are not highlighted and, therefore, the reduction marks (//) are used. A part of WT gag / pol (center) is shown, showing the genes gag (black box) and pol (gray box) followed by the ^r R ^e . The two components that are homologous between WT gag / pol and the vector genome (the RRE and the first 354 bp of gag) are represented in parentheses (they are not drawn to scale). The hypothetical region of recombination between the vector genome and the W ^t gag / pol is shown with connection lines. A part of RI gag / pol (below) is shown showing the codon optimized gag and pol genes (white boxes to distinguish them from the wild type). The reading frame of non-optimized codons between the gag and pol genes is represented in their sequence of natural codons (overlapping black and gray boxes). The primers used for the psi-gag recombination assay are shown with their approximate locations in the constructs. The primers of the first cycle of the PCR are represented by closed arrows and are the pairs 709 and 710 (to detect the recombinant psi-gag) or 709 and 378 (to detect the genome of the integrated vector). The expected amplicon for the vector integrated with primer pairs 709 and 378 is 1697 bp. Nested PCR primers are represented by open arrows and are pairs 863 and 835 (to detect recombinant psi-gag with WT gag / pol) or 863 and 864 (to detect recombination of psi-gag with RI gag / pol) . The expected amplicon with any of the pairs of recombinant primers psi-gag is 937 bp.

Figure 13: 293T cells were transfected with WT gag / pol or RI gag / pol plasmids in the presence of the plasmid Rev (+) or empty main chain plasmid (-). Twenty-four hours later, the cells were lysed and the expression of Gag proteins was analyzed using anti-p24 antibody. The unprocessed Gag protein (p55) and p24 are indicated by arrows. The lysate of non-transfected cells was included as control. Equal loading of the wells was confirmed with anti-actin antibodies.

Figure 14A: the vector encoding GFP was produced with two amounts of plasmids (6 | jg or 3 | jg) of WT gag / pol or RI gag / pol. The resulting p24 levels in the supernatant liquid of the vector are shown in the table. Figure 14B: 293T cells were transduced with 2-fold dilutions of vectors produced in 14A and the expression of GFP was analyzed after 2 days. The UFG / ml were calculated and plotted on the y-axis. The amount of jg of the gag / pol plasmid used to make the vector (6 jg or 3 jg) is plotted on the x axis. WT gag / pol and RI gag / pol are shown in black or white columns respectively.

Figure 15: C57BL / 6 mice were immunized with the indicated doses (2 * 107, 1 * 108, or 5 * 108 TU) of LV encoding the full-length OVA or HBSS vehicle alone. The LVs were of competent integration (INT +) or of poor integration (INT-) and were generated with WT or RI gag / pol, as indicated. On day 12 after immunization, the percentage of splenic CD8 T cells specific for OVA ²⁵⁷ was measured by ICS for IFN- ^and after restimulation of the peptide ex vivo.

Figure 16: C57BL / 6 mice (5 per group) were immunized with 5 * 108 TU of LV integration-deficient (INT-) generated with WT or RI gag / pol encoding a polyepitope antigen (LV1b) containing the cell epitope T CD8 OVA ²⁵⁷ restricted by H-2b and then stimulated on day 28 after immunization with 1 * 107 TCID ⁵⁰ virus of wild-type WR strain vaccine (VV-WT), WR strain recombinant OVA vaccine virus (rVV-OVA), or left unstimulated. On day 33 (day 5 after stimulation) the viral load in the ovaries of each animal was measured by TCID ⁵⁰ assay.

Figure 17A: 293T cells were transduced with the vectors WT gag / pol or RI gag / pol. Two days later, the genomic DNA was isolated and the proviral DNA integrated by PCR was analyzed using the pairs of primers 709 and 378 that bind within the genome of the vector as outlined in FIG. 12. The PCR products were visualized in FIG. a 1% agarose gel. The amplicon size of the vector genome is indicated by an arrow. Mold control was not included in the PCR and the gel. Figure 17B: Progesteric psi-gag recombinants from figure 17A was analyzed in the genomic DNA with the first cycle of PCR followed by a nested PCR. The first PCR cycle used the pairs of primers 709 and 710 that bind in the vector genome and the reading frame within gag / pol respectively. 1 μl of the first cycle PCR product was used as an undiluted template (1: 1), or with 1: 100 or 1: 1000 dilutions in a nested PCR. The primer pairs for the nested PCR were 863 and 835, or 863 and 864. The first pair (863, 835) binds to the vector genome and to WT gag / pol respectively. The last pair (863, 864) binds to the vector genome and to RI gag / pol. All primers and their binding sites are schematized in Figure 12. Only mold controls were included. The resulting nested PCR products were visualized on a 1% agarose gel. The size of the amplicon of the recombinant psi-gag is shown with an arrow. Figure 17C: The nested PCR band for WT gag / pol (the bright band below 1 kb) was cloned into a TOPO-TA vector and sequenced. The sequence of the recombinant band WT gag / pol psi-gag is shown with regions labeled for alignment with the vector genome, partial gag or WT gag / pol. The double bars (//) and the dots indicate sequences that are not shown for simplicity. The numbers in the sequence indicate the position of the nucleotide in the amplicon. Base pairs 1-414 of the recombinant psi-gag are aligned with the genome of the vector, while the base pairs 120-937 are aligned with the WT gag / pol . For reference, the partial sequence of gag is bp 76-439 of the recombinant. Figure 17D: The nested PCR band for RI gag / pol (weak band between 1 kb and 3 kb) was cloned into a TOPO-TA vector and sequenced. The sequence of the RI gag / pol recombinant band is shown with labeled regions for alignment with the vector genome or RI gag / pol . The points indicate sequences that are not shown for simplicity. The numbers in the sequence indicate the position of the nucleotide in the amplicon. The 1 1253 base pairs of the recombinant RI are aligned with the vector genome, while the 1254-1329 bps are aligned with the RI gag / pol . For reference, the partial sequence of gag is bp 76-439 of the recombinant. The complete sequence and alignment are shown in (Supplementary Figure S1B).

Figure 18: HT1080 cells and HT1080 cells stably expressing (A) human DC-SIGN (HT1080 huDC-SIGN), (B) mouse SIGNR1 (HT1080 mSIGNR1), (C) mouse SIGNR3 (HT1080 mSIGNR3) or ( D) mouse SIGNR5 (HT1080 mSIGNR5), were incubated with a pseudotyped VSV-G vector of concentrated deficient integration (1,000 times) encoding GFP, or pseudotyped vectors ID-VP02 encoding GFP that occurred in the absence or presence of kifunensine (Kifu)

Figure 19A: The expression of SIGNR1 and SIGNR5 in spleen cells and lymphatic system was analyzed in individual live cell events. The control staining pattern lacking specific antibodies against SIGNR1 or SIGNR5 is shown. Figure 19B: the individual live spleen and lymph node cell events were subdivided into B cells (labeled B220 + TCRp-, R4), T cells (labeled TCRp +, B220 ', R5) and DC (B220' TCRp 'MHC-II + CD11chi , R7 labeled) and subsequently the expression of SIGNR5 and SIGNR ¹ was analyzed. For all subsets, the frequencies of positive events were <0.00 for negative control stains lacking specific antibodies for SIGNR1 and SIGNR5.

Figure 20: The phenotype of cells expressing GFP in draining lymph nodes was analyzed after subcutaneous injection with ID-VP02 encoding GFP by flow cytometry. Female BALB / c mice (15 per group) were injected subcutaneously into the paw pad of the 3x1010 genomes of ID-VP02 encoding GFP, control ID-VP02 encoding a non-fluorescent protein, or left untreated. Four days later, the popliteal and cervical lymph nodes were grouped separately into 5 mice (3 groups per treatment group) and the presence of cells expressing GFP was analyzed. (A) The expression of GFP was analyzed in individual, live events of the popliteal (draining) or cervical (non-draining) lymph nodes. The cells of the popliteal lymph nodes of ID-VP02 without previous treatment or negative control served as negative controls. (B) The frequency of CD11c and MHC-II is shown in GFP + events of the popliteal lymph nodes of mice injected with ID-VP02 encoding GFP, as overlapping black points on the total of events B220-TCRp-, which they are shown in gray, as a reference. (C) Expression of SIGNR1 in GFP + CD11c + MHC-II + events, shown with (left panel) and without (right panel) inclusion of specific antibody for SIGNR1. The regulation statistics are the average value of three biological repeats. The values in the panel (A) are the number of positive events per 1 * 106 cells, while all other regulation values are percentages.

Figure 21: C57BL / 6 mice (3 per group) received a single subcutaneous injection of 3 * 1010 genomes of ID-VP02 at the base of the tail. The biodistribution, determined by the presence of reverse transcription vector DNA, of ID-VP02 in the indicated tissues was evaluated by quantitative PCR at 1, 4, 8, 21 or 42 days after injection. The inguinal and cervical lymph nodes are indicated as draining and non-draining, respectively. The input DNA was normalized to 200 ng. The limit of quantification (LOQ) of 10 copies per reaction was defined by the lowest copy number in the reference curve. Samples that were not amplified in 40 cycles of qPCR were designated as undetected (ND).

Figure 22: (A) C57BL / 6 mice were immunized with the indicated doses (vector genomes) of ID-VP02 encoding the full-length OVA vehicle or HBSS alone. On day 12 after immunization, the percentage of splenic CD8 T cells specific for OVA ²⁵⁷ was measured by ICS. (B) The kinetics of the response of primary and secondary CD8 T cells to ID-VP02 encoding the OVA was determined by immunization of mice (5 per group) with 1 x 1010 genomes of ID-VP02 in a sensitization regimen- reinforcement with a 35-day interval and analysis of splenic CD8 T cell responses at the indicated measurement times. Immunizations were staggered so that all groups were analyzed by ICS on the same day. (C) Representative intracellular IFN- ^y , TNF-α, and IL-2, and surface CD107a staining on viable CD8 T cells after restimulation with peptide. (D) Frequency of CD8 T cells expressing combinations of IFN- ^y , TNF-a, IL-2, and CD107a around the maximum and posterior contraction of primary and secondary responses. The insignificant numbers of CD8 T cells that were IFN- ^and negative expressed any other effector molecule. (E) The effector / memory phenotype of pentamer CD8 + T cells CD44hi H-2Kb-OVA257 was evaluated by staining with CD127 and KLRG1 at the indicated measurement times.

Figure 23: (A) Experimental program: C57BL / 6 mice (5 per group) were immunized with 5 x ^{1 0 10} , 1 x ^{1 0 10} or 2 x 109 vector genomes of ID-VP02 encoding a polyepitope antigen (LV1b) containing the CD8 T-cell epitopes H-2b-restricted OVA ²⁵⁷ and LCMV GP ³³ and then stimulated on day 35 after immunization with 1x107 TCID50 wild type WR vaccine virus (VV-WT), virus Recombinant OVA vaccine strain WR (rVV-OVA), or left unstimulated. On day 40 (day 5 after stimulation) the responses of splenic CD8 T cells and the viral load in the ovaries were measured. (B) OVA ²⁵⁷ - and LCMV GP ³³ specific CD8 T cell responses were measured by intracellular IFN- ^y and TNF-α staining after restimulation with ex vivo peptide. Graphs of points representative of the cytokine profile of CD8 T cells are shown. (C) Frequency of CD8 T-cells IFN-Y + specific for OVA ²⁵⁷ for each animal. (D) Viral load (measured by the TCID ⁵⁰ test) within the ovaries of each animal.

Figure 24: (A) BALB / c mice (5 per group) were immunized with the indicated doses, with vector genomes, of ID-VP02 encoding AH1A5, a heteroclite mutant of the endogenous tumor rejection CT26 AH1 epitope, bound to OVA (OVA- AH1A5) or HBSS vehicle only. On day 12 after immunization, the percentage of splenic CD8 T cells specific for AH1A5 or AH1 by ICS was measured. (B) Twelve days after immunization, a 1: 1: 1 mixture of dye-labeled target cells each pulsed with AH1, AH ¹ A ⁵ or a control peptide was intravenously transferred to immunized and untreated mice ( 3 per group). The next day, the spleens were collected and the relative recovery of each population was compared between mice without previous treatment and immunized to calculate the specific death. (C) BALB / c mice (10 per group) were immunized with 4 x 109 genomes of ID-VP02 vectors encoding OVA-AH1A5. Four weeks later, the mice were injected subcutaneously with 8x10 4 CT26 tumor cells on the right flank and the mice were sacrificed when the tumors exceeded 100 mm2. (D) Therapeutic immunization: BALB / c mice (10 per group) were injected subcutaneously with 8 x 10 4 CT26 tumor cells. Four days later, the mice were immunized with 4x109 vector genomes of ID-VP02 which encode OVA-AH1A5 or were left untreated and the mice were sacrificed when the tumors exceeded 100 mm2.

Detailed description

This description relates to methods and materials useful for generating particles of pseudotyped lentiviral vectors that efficiently bind and productively infect cells expressing DC-SIGN (eg, dendritic cells). The methods and materials in this disclosure relate to the unexpected discovery that the combination of a Vpx protein in a lentiviral vector particle with alphavirus glycoproteins (eg, Sindbis glycoproteins) highly mannosylated (e.g. culture of virus packaging cells in the presence of kifunensine) in the shell results in particles of lentiviral vectors that infect non-dividing cells expressing DC-SIGN (eg, dendritic cells) significantly more effectively than the cells. particles of lentiviral vectors lacking a Vpx protein or highly-mannosylated glycoproteins in the envelope. An advantageous aspect of the description is that the particles of lentiviral vectors pseudotyped with Sindbis virus glycoprotein e2 and produced in the presence of kifunensin, infect dendritic cells significantly more efficiently if the particles also comprise a Vpx protein in the virion, thus allowing the delivery and expression of a sequence of interest (eg, a polynucleotide encoding an antigen) to a dendritic cell.

Definitions

The term "functional fragment" when used in reference to a polypeptide means a polypeptide that is truncated, i.e., one or more amino acids are missing from the N-terminus or C-terminus, and that it retains the desired activity. When used in reference to a Vpx protein, "functional fragment" means a fragment that retains the ability to inhibit the activity of SAMHD1. The term can be applied analogously to polynucleotides that are truncated.

The term "variant" when used in reference to a polypeptide means a polypeptide having one or more substitutions, deletions or insertions with respect to an original polypeptide. In some contexts, the variant is one that retains the desired activity. When used in reference to a Vpx protein, "functional variant" means a variant that retains the ability to inhibit the activity of SAMHD1. The expression can be applied analogously to polynucleotides having one or more substitutions, deletions or insertions with respect to an original polynucleotide.

As used herein, the term "conservative amino acid substitution" is the substitution of one amino acid with another amino acid having similar properties, e.g. eg, size, charge, hydrophobicity, hydrophilicity and / or aromaticity, and includes exchanges as indicated below:

Original Example

Wing (A) val; leu; ile

Arg (R) lys; gln; asn

Asn (N) gln; his; asp, lys; gln

Asp (D) glu; asn

Cys (C) be; to

Gln (Q) asn; glu

Glu (E) asp; gln

Gly (G) wing

His (H) asn; gln; lys; arg

Ile (I) leu; val; met; to;

phe; norleucine

Leu (L) norleucine; ile; val;

met; to; phe

Lys (K) arg; gln; asn

Met (M) leu; phe; ile

Phe (F) leu; val; ile; to; tyr

Pro (P) wing

Ser (S) thr

Thr (T) be

Trp (W) tyr; phe

Tyr (Y) trp; phe; thr; be

Val (V) ile; leu; met; phe;

to; norleucine

The amino acid residues that share common properties of the side chain are often grouped as follows:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acid: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence the orientation of the chain: gly, pro; Y

(6) aromatic: trp, tyr, phe.

As used herein, the terms "poor integration" and "defective integration" are used interchangeably. A viral integrative deficient vector is one that is at least 10 times (preferably, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times, at least 300 times, at least 350 times, at least 400 times, at least 450 times, at least 500 times, at least 550 times, at least 600 times, at least 650 times, at least 700 times, at least 750 times, at least 800 times, at least 850 times, at least 900 times, at least 950 times, or at least 1000 times) less effective in integration than a competent integration viral vector. For example, poor integration can mean at least about 20 times to about 100 times less effective in integration than a viral vector that has not been modified to be defective. The integration can be measured by any method known in the art, e.g. eg, by detection by PCR of integration events near an Alu element.

A "pseudotyped" lentivirus is a lentiviral particle that has one or more envelope glycoproteins that are encoded by a virus that is distinct from the lentiviral genome. The envelope glycoprotein can be modified, mutated or designed as described herein.

As used herein, the term "exogenous antigen" refers to an antigen that has been genetically modified to be expressed in the lentiviral vectors described herein. Accordingly, the term explicitly includes antigens derived from HIV that have been genetically engineered to be expressed in the lentiviral vectors described herein.

As used herein, a Sindbis E2 glycoprotein that "preferentially binds dendritic cells expressing DC-SIGN" is a glycoprotein that binds to dendritic cells that express DC-SIGN more efficiently than non-expressing cells DC-SIGN.

The envelope protein of the Sindbis virus contains four glycans bound to N, two in the E2 protein and two in the E1 protein. Two N-glycans of the virus produced in mammalian cells in the absence of a mannosidase I inhibitor have a structure with high mannose content (one glycan bound to N of E2 and one glycan bound to N of E1), while the remaining two They have a complex structure. The two N-glycans of complex structure are exposed on the surface of the envelope protein, while the two structure N-glycans with high mannose content are buried within the trimer center of the envelope proteins. See, Morizono et al., J Virol, 84:14, 6923-34 (2010). Accordingly, typically 50% of the glycans bound to N in a viral particle with a Sindbis virus glycoprotein produced in mammalian cells would have the structure of high mannose content. A "highly mannosylated" viral particle is a particle in which at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% of the glycans bound to N in the viral envelope glycoproteins comprise at least one structure of Man ⁵ , preferably Mang, as measured, for example, by mass spectrometry (see, Crispin et al., JBC 2009).

SAMHD1 inhibitors

Vpx and Vpr

In some or any of the embodiments, the SAMHD1 inhibitor is a Vpx protein or a Vpr protein. Vpx is encoded by HIV-2 virus, SIV of mangabey gray (SIVsm), SIV of mangabey of red beret (SlVrcm) and SIV of macaco (SlVmac), among others. Vpx of HIV-2 and SIV is a protein of 112 amino acids (aa), 18 kDa, and is packaged in the virion in significant quantities through its interaction with the p6 region of the p55gag precursor. Recently it was demonstrated that Vpx inhibits the activity of a restriction factor expressed in human dendritic and myeloid cells, SAMHD ¹ . Laguette et al., Nature, 474, 654-657 (2011). SAMHD1 was identified as the restriction factor that makes human dendritic and myeloid cells largely refractory to HIV-1 infection.

Vpx of SIV and HIV-2 are 83% identical at the amino acid level. See, Goujon et al., J Virol, 82:24, 12335-12345 (2008). Therefore, it can be assumed that the residues that differ between SIV and HIV-2 are not important for the function of Vpx. In addition, others have carried out the Vpx mutational analysis of SIV and HIV-2 and can be used as a guide to generate Vpx variants for use in the materials and methods of this disclosure. See, Goujon et al. The mutants Asn26Ala, Ser52Ala and Ser63Ala / Ser65Ala do not affect the function of Vpx. The elimination of the remaining 11 C-terminal, proline-rich, mutations Ser13Ala, Lys84Ala / Lys85Ala, Thr17Ala, Thr28Ala, Gly86Ala / Cys87Ala, Ser13Ala / Thr17Ala / Thr28Ala, His39Ala, and Tyr66Ala / Tyr68Ala / Tyr71Ala, Trp49Ala / Trp53Ala / Trp56Ala, and Lys68Ala / Lys77Ala cancel the activity of Vpx.

In some or any of the embodiments, the lentiviral vector particles described herein comprise a Vpx protein or a variant thereof. In some or in any of the embodiments, the variant retains the ability to inhibit SAMHD1. In some or any of the embodiments, the variant comprises at least 75%, 80%, 85%, 90% or 95% amino acid sequence identical to SEQ ID NO: 45 (SIVsm Vpx). In some or any of the embodiments, the variant comprises at least 75%, 80%, 85%, 90% or 95% amino acid sequence identical to SEQ ID NO: 46 (SIVrcm Vpx). In some or any of the embodiments, the variant comprises at least 75%, 80%, 85%, 90% or 95% amino acid sequence identical to SEQ ID NO: 44 (SIVmac Vpx). In some or any of the embodiments, the variant comprises at least 75%, 80%, 85%, 90% or 95% amino acid sequence identical to SEQ ID NO: 47 (HIV-2).

The anti-SAMHD1 activity of Vpx has been localized in the 86 N-terminal residues of Vpx. Gramberg et al., J Virol, 84: 3, 1387-1396. Accordingly, in some or any of the embodiments, the functional fragment comprises the inhibitory region of SAMHD1 of Vpx, ie, amino acid residues 1 to 86 of SEQ ID NO: 44.

While Vpx is only present in some lentiviruses, all primate lentiviruses encode a gene closely related to Vpx called Vpr. It is known that Vpr causes the arrest of the cell cycle. Recently, however, it was shown that Vpr proteins isolated from SIVdeb and SIVmus inhibited human SAMHD1. Lim et al., Cell Host & Microbe, 11, 194-204 (2012). Accordingly, in some or any of the embodiments, the lentiviral vector particles described herein comprise a Vpr protein that inhibits SAMHD1 or a variant thereof that retains the ability to inhibit SAMHD1. In some or any of the embodiments, the variant comprises at least 75%, 80%, 85%, 90% or 95% amino acid sequence identical to SEQ ID NO: 48 (SIVdeb Vpr). In some or any of the embodiments, the variant it comprises at least 75%, 80%, 85%, 90% or 95% amino acid sequence identical to SEQ ID NO: 49 (SlVmus Vpr).

The information of the Vpx protein sequence alignments can be used to generate functional variants and functional fragment variants of Vpx, as defined above. Techniques for removing and mutating amino acids are well known in the art. See, Ausubel, FM et al., Current Protocols in Molecular Biology, John Wiley & Sons, (1998), including all supplements up to 2011. In general, to construct functional variants, non-conservative or conservative substitutions or deletions can be introduced in the positions that differ between the viruses that encode a Vpx protein, since these positions tend to be non-conservative substitutions allowed while retaining the function. For positions with amino acid residues conserved through the viruses, the remainder is conserved or conservative substitutions are introduced. The ability of Vpx, Vpr and its variants to inhibit the activity of SAMHD1 is tested according to the methods described herein or known in the art. See, Lim et al., Cell Host & Microbe, 11, 194-204 (2012) and Lahouassa et al., Nature Immunol, 13: 3, 223-229 (2012).

Despite previous reports indicating that Vpx packaging of SIVmac in HIV-1 virions required modification of the p6 region of the Gag protein to resemble pIV of SIVmac (Sunseri et al., J Virol, 86: 6 ( 2012)), the inventors have unexpectedly discovered that SIVmac Vpx is effectively packaged in the pseudotyped HIV-1-based lentiviral vectors described herein without the need to modify p6 or fuse Vpx to Vpr of HIV-1. Accordingly, in some or any of the embodiments, the Vpx protein is not fused to a Vpr protein. Similarly, in some or any of the embodiments, the gag protein in the packaging cell is not modified from its natural sequence.

In some or any of the embodiments, the Vpx protein is fused with the Vpr protein of HIV-1.

Typically, the Vpx protein is packaged in the viral particle. However, in some or any of the embodiments, a gene encoding a Vpx protein is included in the lentiviral genome and is expressed when the viral particle infects a target cell.

Wrapped viral vector

Viruses transmitted by arthropods (Arboviruses) are viruses that are transmitted to a host, such as humans, horses or birds, by an infected arthropod vector, such as a mosquito. Arboviruses are further divided into virus subfamilies, including alphaviruses and flaviviruses, which have a positive polarity single-stranded RNA genome and a glycoprotein-containing envelope. For example, the virus of dengue fever, yellow fever virus and West Nile virus belong to the flavivirus family, and the Sindbis virus, Semliki forest virus and Venezuelan equine encephalitis virus are members of the alphavirus family (Wang et al., J. Virol. 66, 4992 (1992)). The Sindbis virus envelope includes two transmembrane glycoproteins (Mukhopadhyay et al., Nature Rev. Microbio , 3, 13 (2005)): E1, which is believed to be responsible for the fusion, and E2, which is believed to be responsible for the binding mobile. Sindbis virus envelope glycoproteins are known to pseudotype other retroviruses, including oncorretrovirus and lentivirus.

The Sindbis virus and other alphavirus envelopes are incorporated into the lipid bilayer of the viral particle membrane, and typically include multiple copies of two glycoproteins, E1 and E2. Each glycoprotein has regions that extend across the membrane; E2 has a cytoplasmic domain of approximately 33 residues, while the cytoplasmic tail of E1 is very short (approximately 2 residues). Both E1 and E2 have palmitic acids bound in or near the regions that extend across the membrane. E2 is initially synthesized as a precursor protein that is cleaved by furin or another serine proteinase dependent on Ca2 + in E2 and in a small glycoprotein called E3. Located between the sequences that encode E2 and E1 is a sequence that encodes a protein called 6K. E3 and 6K are signal sequences that serve to translocate glycoproteins E2 and E1, respectively, to the membrane. In the Sindbis virus genome, the coding region for the Sindbis envelope proteins includes the sequence encoding E3, E2, 6K and E1. As used herein, the "envelope" of an arbovirus virus includes at least E2, and may also include E1, 6K and E3. An example sequence of envelope glycoproteins of the Sindbis virus, strain HR, is presented as SEQ ID NO: 17 (polyprotein E3, E2, 6K and E1). Sequences of the envelope glycoproteins for other arboviruses can be found, e.g. ex., in GenBank. For example, the sequence encoding dengue virus glycoproteins can be found in accession no. GQ252677 (among others in GenBank) and in the database of virus variations in the NCBI (GenBank accession no. database of virus variations refer to the envelope glycoprotein sequences) and the sequence encoding the Venezuelan equine encephalitis virus in accession number NP 040824 (which refers to envelope glycoprotein sequences) .

It is understood that references to a "E2 glycoprotein residue number" (such as residue 160, 70, 76 or 159) are defined by reference to the amino acid sequence of the protein of SEQ ID NO: 18, which is the E2 glycoprotein of the Sindbis strain HR (ie, "Sindbis HR reference strain"). For example, if a variant of Sindbis glycoprotein E2 has been produced in which the residue 70 has been deleted from SEQ ID NO: 18, then "residue 160" will refer to the actual residue 159 of said variant. Analogous positions in other alphaviruses can be determined by alignments that maximize homology.

The use of the terms "ligated", "binding", "targeting" and the like are used interchangeably and are not intended to indicate a mechanism of the interaction between the envelope glycoprotein of the Sindbis virus and a cellular component. DC-SIGN (ICAM-3 (intercellular adhesion molecule 3) not associated with integrin, specific for dendritic cells, also known as CD209) is a type C lectin receptor capable of rapid binding and endocytosis of materials (Geijtenbeek, TB, et al. Annu., Rev. Immunol., 22: 33-54, 2004). It seems that E2 directs viruses to dendritic cells through DC-SIGN. As shown herein, cells expressing DC-SIGN are transduced by viral vector particles pseudotyped with E2 from Sindbis virus best (at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times better) than isogenic cells that do not express DC-SIGN. The mechanism of how the E2 glycoprotein facilitates viral infection appears to involve DC-SIGN, possibly through direct binding to DC-SIGN or causing a change in conformation or some other mechanism. Regardless of the actual mechanism, E2 targeting is preferential for cells expressing DC-SIGN, in particular, dendritic cells.

Sindbis virus also binds to cells by heparan sulfate (Klimstra et al., J Virol 72: 7357, 1998, Burmes and Griffin, J Virol 72: 7349, 1998). Because heparan sulfate and other glycosaminoglycans on the cell surface are found on the surface of most cell types, it is desirable to reduce the interaction between heparan sulfate and the glycoproteins of the Sindbis envelope. This can be achieved by decreasing the binding of the Sindbis virus envelope to heparan sulfate or by increasing the binding, e.g. eg, increasing the avidity, of the Sindbis virus envelope to the dendritic cells or both. As a result, non-specific binding to other molecules, which can be expressed by other cell types, is reduced and can occur even if the shell is specific for DC-SIGN, and the enhanced specificity can serve to avoid unwanted side effects, such as side effects that can reduce the desired immune response, or side effects associated with transduction outside the target of other cell types. Alternatively or in addition to the advantages of relatively specific transduction of cells expressing DC-SIGN, viral particles pseudotyped with the E2 glycoprotein of the Sindbis virus envelope may offer other advantages over viral particles pseudotyped with glycoproteins such as VSVG. Examples of such advantages include reduction of complement-mediated lysis and / or reduction of targeting of neuronal cells, and both are believed to be associated with the administration of viral particles pseudotyped with VSV-G.

In different embodiments, the lentiviral vector particles described herein specifically bind to cells that express DC-SIGN and have a reduced or ablated binding to heparan sulfate. That is, an E2 glycoprotein of the Sindbis virus envelope can be modified to preferentially target the virus to dendritic cells expressing DC-SIGN with respect to other cell types. Based on the information obtained from structural studies and molecular modeling among other studies, the variant sequences of envelope proteins, especially glycoproteins E2 and E1, are designed and generated in such a way that the glycoproteins maintain their functions as envelope proteins, but have the desired binding, avidity or binding level specificity. Candidate variant sequences can be created for each glycoprotein and assayed using the methods described below, or other methods known in the art, to identify envelope glycoproteins with the most desirable characteristics.

Certain Sindbis E2 variant sequences have at least one amino acid alteration in residue 160 as compared to SEQ ID NO: 1 or SEQ ID NO: 18 (Sindbis HR E2 reference sequence). The residue 160 is removed or changed to an amino acid other than glutamic acid. An alteration is most commonly a substitution of at least one amino acid, but alternatively it may be an addition or deletion of one or more amino acids. Preferably, any additional amino acids are few in number and do not comprise an antigenic epitope (e.g., hemagglutinin marker sequence), which may compromise safety. When there are two or more alterations, both may be of the same type (eg, substitution) or of different types (eg, a substitution and a deletion). Multiple alterations may be scattered or located contiguously in the protein sequence.

In some embodiments, the variant sequences comprise at least one amino acid alteration in the region from about residue 50 to about E2 180 residue of the Sindbis virus (SEQ ID NO: 18). Within this region there are amino acids that are involved in binding to heparan sulfate. By reducing the net positive charge of E2, the electrostatic interaction with heparan sulfate can be reduced, resulting in a lower binding to heparan sulfate. The candidate positively charged amino acids in this region include lysines at residues 63, 70, 76, 84, 97, 104, 129, 131, 133, 139, 148, 149, 159 and arginine at residues 65, 92, 128, 137, 157, 170, 172 (Bear et al., Virology 347: 183-190, 2006) of SEQ ID NO: 18. At least several of these amino acids are directly involved in the binding of E2 to heparan sulfate. The net positive charge can be reduced by removing lysine or arginine or replacing lysine or arginine with a neutral or negatively charged amino acid. For example, one or more of these lysines and arginines can be replaced by glutamic or aspartic acid. Certain embodiments have at least one substitution of lysine 70, 76 or 159. In cases where E2 is expressed as a polyprotein with E3, the lysine located adjacent to the natural E3 / E2 cleavage site is maintained, i.e. the sequence of recognition and the cleavage site are unchanged. Alternatively, the sequence of the cleavage site of the native endopeptidase is replaced by a recognition sequence for a different endopeptidase.

Some E2 variants of the Sindbis virus are also modified in a way that has a positive impact on the binding to the dendritic cells. Alteration of the glutamic acid found in residue 160 in the HR reference sequence (SEQ ID NO: 18) may improve binding to dendritic cells (see, Gardner et al., J Virol 74, 11849, 2000). In some variants alterations are found, such as a deletion of residue 160 or substitution of residue 160. In particular variants, an uncharged amino acid is replaced by Glu, in other variants, a non-acidic amino acid is replaced by Glu. Normally, Glu160 is replaced by one of the small or aliphatic amino acids, which include glycine, alanine, valine, leucine or isoleucine.

Other variants comprise two or more (eg, 3, 4 or 5) amino acid alterations. Typically, in these variants, one of the alterations is Glu160 and the or the remaining alterations are changes of one or more of the lysins and arginines in the region encompassing the residues from about 50 to about 180. Some of the variants comprise an alteration from Glu160 to non-acidic residue or elimination and one or more alterations of lysine 70, lysine 76 or lysine 159 with a non-basic amino acid. Some specific variants comprise a Glu160 to Gly, Lys 70 to Glu and Lys 159 to Glu; one Glu 160 to Gly, Lys 70, 76 and 159 to Glu; an elimination of Glu 160 and Lys 70 and 159 to Glu; and an elimination of Glu 160 and Lys 70, 76 and 159 to Glu. In some embodiments, the E2 variant is between 80 and 100% (eg, 82%, 85%, 87%, 90%, 92%, 95%, 97% or 99%) identical to any one of the SEQ ID NO: 30 -43 [SINvar1-14 E2].

In certain cases, the E2 protein is first expressed as a polyprotein in fusion with at least E3 or in fusion with a leader sequence. Regardless of whether the leader sequence is E3 or another sequence, E2 in the viral envelope must lack E3 or another leader sequence. In other words, E2 is preferably not an E3 / E2 fusion protein. In certain embodiments, E2 is expressed as part of the polyprotein E3-E2-6K-E1. In these embodiments, the polyprotein is between 80 and 100% (eg, 82%, 85%, 87%, 90%, 92%, 95%, 97% or 99%) identical to any of SEQ ID NO. : 3-16 [polyprotein SINvar1-14]. The Sindbis virus naturally expresses E2 as part of a polyprotein and the binding regions for E3 / E2, E2 / 6K, and 6K / E1 have recognized and cleaved sequences by endopeptidases. Typically, the E3 / E2 junction is cleaved by furin or a serine-like serine endopeptidase between residues 65 and 66. Furin has specificity for paired arginine residues that are separated by two amino acids. To maintain the cleavage of E3 / E2 by furin, residues 62-66 (RSKRS; SEQ ID NO: 26) must maintain the two arginine residues with separation of two amino acids and the rest of serine. Alternatively, a different cleavage sequence can be used in place of the furin cleavage sequence of E3 / E2 or any of the other cleavage sequences. Recognition and cleavage sites for endopeptidases can be incorporated, including, without limitation, aspartic endopeptidases (eg, cathepsin D, chymosin, HIV protease), cysteine endopeptidases (bromelains, papain, calpain), metalloendopeptidases (e.g. e.g., collagenase, thermolysin), serine endopeptidases (e.g., chymotrypsin, factor IXa, factor X, thrombin, trypsin), streptokinase. The recognition sequences and cleavage sites of these enzymes are well known.

E2 amino acids of Sindbis virus can also be altered, other than those already mentioned. In general, a variant sequence of E2 will have at least 80% amino acid sequence identity with the E2 reference sequence, or can have at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 95%, or at least 98% sequence identity. In some embodiments, a variant sequence of E2 has at least 80% amino acid sequence identity with SEQ ID NO: 30 [SINvar1], or it can have at least 82%, at least 85%, at least 87%, at minus 90%, at least 92%, at least 95%, or at least 98% sequence identity. In some embodiments, a variant E2 sequence has at least 80% amino acid sequence identity with SEQ ID NO: 31 [SINvar2], or it can be at least 82%, at least 85%, at least 87%, at minus 90%, at least 92%, at least 95%, or at least 98% sequence identity. In some embodiments, a variant E2 sequence has at least 80% amino acid sequence identity with SEQ ID NO: 32 [SINvar3], or it can be at least 82%, at least 85%, at least 87%, at minus 90%, at least 92%, at least 95%, or at least 98% sequence identity.

The variant glycoprotein should have biological function, such as the ability to facilitate the infection of dendritic cells by a viral particle having a shell comprising E2. The experiments have identified regions of envelope glycoproteins that appear to play an important role in various aspects of viral assembly, cell surface binding, and infection. When making variants, the following information can be used as a guide. The cytoplasmic tail of E2 - approximately residues 408 to 415 - is important for the assembly of the virus (West et al., J Virol 80: 4458-4468, 2006). Other regions are involved in the formation of the secondary structure (approximately residues 33-53); and involved in the transport and stability of proteins (approximately residues 86-119) (Navaratmarajah et al., J Virol 363: 124-147, 2007). The variant can retain the hydrophobic character of a region extending across the membrane, approximately residues 370-380. The variant can retain one or both glycosylation sites attached to N NIT residues (residues 196 198) and NFT (residues 318-320) and can retain one or more of the palmitoylated sites (C-396, ^c 416 and C417) (Strauss and Strauss Microbiol Rev 58, 491-562, 1994, page 499-509 incorporated). On the other hand, many regions of E2 can be altered without damaging event. For example, insertions of transposons in many different places in E2 still resulted in a viable virus (Navaratmarajah, ibidem).

In certain embodiments, a marker peptide can be incorporated into E3, 6K or E1 proteins. For some purposes, a label can be incorporated into E2, but a label for use in a product for administration to human patients is not desirable. A marker peptide, which is a short sequence (e.g., 5-30 amino acids), to facilitate the detection of the expression of the envelope and its presence in viral particles. For detection purposes, a marker sequence will typically be detectable by antibodies or chemicals. Another use for a marker is to facilitate the purification of viral particles. A substrate containing a binding partner for the marker can be used to absorb the virus. Elution of the virus can be achieved by treatment with a motif that displaces the marker from the binding partner or when the marker sequence is in connection with a cleavable sequence, treatment with the appropriate endopeptidase will conveniently allow virus release. (See, for example, Qiagen catalog, Factor Xa Protease System). The elimination of the marker peptide in general is desirable for safety purposes of the use of virus particles in animal subjects. If the marker is not removed, an immune response against the marker may occur.

Suitable markers include, without limitation, FLAG (DYKDDDDK) (SEQ ID NO: 28) (U.S. Patent No. 4,703,004, for which antibodies are available, chitin binding protein, binding to maltose, glutathione-S-transferase, poly (His) (U.S. Patent No. 4,569,794), thioredoxin, HA (hemagglutinin) -marker, etc. Poly (His) can be adsorbed to media of affinity containing bound metal ions, eg, nickel or cobalt, and eluting with a medium at low pH.

Viral particles can be evaluated to determine the specificity of the envelope glycoprotein incorporated in the virus that targets the dendritic cells. For example, a mixed population of bone marrow cells from a subject can be obtained and cultured in vitro. Alternatively, isogenic cell lines expressing or not expressing DC-SIGN can be obtained and used. The recombinant virus can be administered to the mixed population of bone marrow cells or isogenic cell lines, and the expression of a reporter gene incorporated into the virus in the cultured cells can be analyzed. Certain embodiments may use a limiting dilution analysis, in which the mixed population of cells is divided into separate parts, which are then incubated separately with decreasing amounts of virus (eg, 2 times, 5 times, 10 times less virus in each part). In some embodiments, at least about 50%, more preferably at least about 60%, 70%, 80% or 90%, even more preferably at least about 95% of the infected cells in the mixed cell population are dendritic cells expressing DC-SIGN. In certain embodiments, the ratio of infected dendritic cells to infected non-dendritic cells (or cells not expressing DC-SIGN) is at least about 2: 1, at least about 3: 1, at least about 4: 1, at least about 5: 1, at least about 6: 1, at least about 7: 1, at least about 8: 1, at least about 9: 1, at least about 10: 1, at least about 20: 1, at least about 30 : 1, at least about 40: 1, at least about 50: 1, at least about 100: 1, at least about 200: 1, at least about 500: 1, at least about 1000: 1, at least about 5000: 1, at least about 10,000: 1, or more. For limiting dilution, higher selectivity is typically observed at higher dilutions (i.e., smaller amounts) of input virus.

The activity of the pseudotyped viral particles can be determined by any of a variety of techniques. For example, a preferred method for measuring the effectiveness of infectivity (UI, infectious units) is to deliver viral particles to cells and measure the expression of a product encoded in the vector genome. Any product that can be analyzed can be used. A convenient type of product is a fluorescent protein, such as green fluorescent protein (GFP). Other products that can be used include proteins expressed on a cell surface (eg, detection by antibody binding), enzymes and the like. If the product is an antigen and the cells are dendritic cells, the infectivity / activity can be assessed by determining an immune response. In addition, side effects can be determined in a mammal. The ability to specifically target dendritic cells can also be directly assayed, for example, in cell culture as described below.

Viral particles can also be prepared and tested for their selectivity and / or their ability to facilitate penetration of the target cell membrane. Viral particles that have a shell with unmodified glycoproteins can be used as controls for comparison. Briefly, cells expressing a receptor for a envelope glycoprotein are infected by the virus using a standard infection assay. After a specific time, for example, 48 hours after infection, the cells can be harvested and the percentage of cells infected with the virus can be determined by flow cytometry, for example. The selectivity can be qualified by calculating the percentage of cells infected by viruses. Similarly, the effect of a variant envelope glycoprotein on the viral titer can be quantified by dividing the percentage of infected cells by virus comprising a variant envelope between the percentage of virus-infected cells comprising the corresponding envelope glycoprotein type. natural (not modified). A particularly suitable variant will have the best combination of selectivity and infectious titer. Once a variant is selected, viral concentration assays can be performed to confirm that these viruses can be concentrated without compromising activity. The viral supernatants are collected and concentrated by ultracentrifugation. The titers of the viruses can be determined by limited dilution of virus stock solution and infection of the cells expressing the envelope glycoprotein receptor, by measuring the expression of a product expressed by the viruses as described above.

The entry of a lentiviral vector particle into a target cell is another type of activity evaluation. The fusion protein BlaM-Vpr (beta-lactamase Vpr) has been used to evaluate viral penetration of HIV-1; a fusion of BlaM and a glycoprotein of the Sindbis virus envelope, such as E1 or a protein of E2 / E1 fusion to evaluate the effectiveness of a shell protein to facilitate fusion and penetration into a target cell. Viral particles can be prepared, for example, by transient transfection of packaging cells with one or more vectors comprising the viral elements, BlaM-Vpr and the envelope of the variant of interest (and an affinity molecule if appropriate). The resulting viruses can be used to infect cells expressing a molecule, the targeting molecule (or affinity molecule) binds specifically in the absence or presence of the free binding inhibitor (such as an antibody). The cells can then be washed with an independent CO ² medium and loaded with CCF2 dye (Aurora Bioscience). After incubation at room temperature to allow the cleavage reaction to complete, the cells can be fixed by paraformaldehyde and analyzed by flow cytometry and microscopy. The presence of blue cells indicates the penetration of virus into the cytoplasm; fewer blue cells would be expected when the blocking antibody is added (Cavrois et al, Nat Biotechnol 20: 1151-1154, 2002).

To investigate whether penetration depends on a low pH, and to identify envelope glycoproteins with the desired pH dependence, NH ⁴ Cl or another compound that alters the pH at the infection stage can be added (NH ⁴ Cl will neutralize the acid compartments of the endosomes). In the case of NH ⁴ Cl, the disappearance of blue cells will indicate that the penetration of the viruses depends on the low pH. In addition, to confirm that the activity depends on the pH, lysomotropic agents, such as ammonium chloride, chloroquine, concanamycin, bafilomycin A1, monensin, nigericin, etc., may be added to the incubation buffer. These agents raise the pH within the endosomal compartments (eg, Drose and Altendorf, J. Exp. Biol. 200, 1-8, 1997). The inhibitory effect of these agents will highlight the role of pH in the fusion and entry of viruses. The different input kinetics between viruses that have different fusogenic molecules can be compared and the most suitable for a particular application can be selected.

PCR-based input assays can be used to control reverse transcription and measure the kinetics of viral DNA synthesis as an indication of the kinetics of virus entry. For example, viral particles comprising a particular envelope protein molecule are incubated with target cells, such as 293T cells, DC or any other cell that is designed to express, or naturally express, the binding partner. adequate (receptor) for the envelope protein molecule. Immediately, or after an increase in time (to allow the infection to occur), unbound viruses are removed and viral nucleic acids are analyzed in aliquots of the cells. The DNA is extracted from these aliquots and subjected to analysis by amplification, generally in a semiquantitative assay, primed with specific LTR primers. The appearance of LTR-specific DNA products indicates the success of virus entry.

Lentiviral vector genome

The viral vector particle comprises a genome, comprising at least one sequence (eg, 1, 2, 3, 4 or 5) of interest. Other sequences may be included, such as sequences that allow the genome to be packaged in the viral particle and sequences that promote the expression of the / sequences of interest after transduction of the target cell. The genome can be obtained from any of a large number of vectors based on suitable available lentiviral genomes, including those identified for human gene therapy applications, such as those described by Pfeifer and Verma (Annu., Rev. Genomics Hum. Genet. 177-211, 2001). For the sake of simplicity, the genome is also called the "viral vector genome" or "vector genome". Main chain

Suitable lentiviral vector genomes include those based on human immunodeficiency virus (HIV-1), HIV-2, feline immunodeficiency virus (FIV), equine infectious anemia virus, simian immunodeficiency virus (SIV) and maedi / visna virus. A desirable feature of lentiviruses is that they can infect both dividing cells and non-dividing cells, it is not necessary for the target cells to divide (or stimulate the target cells to divide). In general, the genome and the envelope glycoproteins will be based on different viruses, so that the resulting viral vector particle is pseudotyped. The safety features of the vector genome are conveniently incorporated. Security features include auto-activating LTR and a genome that is not integrated.

In some exemplary embodiments, the genome of the viral vector comprises sequences from a lentivirus genome, such as the HIV-1 genome or the SIV genome. The viral genome construct can comprise sequences of the 5 'and 3' LTRs of a lentivirus, and in particular can comprise the R and U5 sequences of the 5 'LTR of a lentivirus and an inactivated or autoinctivating LTR 3' of a lentivirus. The LTR sequences can be LTR sequences of any lentivirus of any species. For example, they can be LTR sequences of HIV, SIV, FIV or BIV. Typically, the LTR sequences are HIV LTR sequences.

The vector genome may comprise an inactivated or auto-inactivating 3 'LTR (Zufferey et al., J Virol 72: 9873, 1998, Miyoshi et al., J Virol 72: 8150, 1998, US2010 / 0323403). A self-inactivating vector generally has a deletion of the 3 'long terminal repeat (LTR) promoter and promoter sequences, which is copied into the 5' LTR during vector integration. In one case, the U3 element of the 3 'LTR contains a deletion of its enhancer sequence, the polypurine tract (PPT), the TATA box, the Sp1 and NF-kappa B sites. As a result of the 3' LTR self-activating, the provirus that is generated after the input and reverse transcription will comprise an LTR 5 'inactivated. The rationale is to improve safety by reducing the risk of vector genome mobilization and the influence of LTR on nearby cellular promoters. The auto-activating LTR 3 'can be constructed by any method known in the art. In various embodiments, the pseudotyped lentiviral vector comprises a lentiviral vector genome comprising the sequence of SEQ ID NO: 23. This vector comprises an elimination of the 3 'PPT and the TATA box. Therefore, the vector is self-inactivating, so that (a) genome transcription of the full-length vector is avoided from reverse transcribed dsDNA vectors in the infected target cell, (b) the risk of insert activation that can occur when a 3 'LTR can function as a promoter after integration, and (c) the removal of extended U3 can be supplemented by additional and redundant safety mechanisms (eg, integrase mutation).

Optionally, the U3 sequence of the lentiviral LTR 5 'can be replaced by a promoter sequence in the viral construct, such as a heterologous promoter sequence. This can increase the virus titer recovered from the packaging cell line. An enhancer sequence may also be included. Any combination of enhancer / promoter that enhances expression of the viral RNA genome in the packaging cell line can be used. In one example, the CMV enhancer / promoter sequence is used (U.S. Patent No. 5,385,839 and U.S. Patent No. 5,168,062).

In certain embodiments, the risk of insertion mutagenesis is minimized by constructing the genome of the lentiviral vector so that it is of defective integration. A variety of approaches can be pursued to produce a non-integrating vector genome. These approaches involve designing a mutation (s) in the integrase enzyme component of the pol gene , so that it encodes a protein with an inactive integrase. The vector genome itself can be modified to avoid integration, for example, by mutating or deleting one or both of the binding sites, or by making the 3 'LTR-proximal non-functional polypurine tract (PPT) by removal or modification. In addition, non-genetic procedures are available; these include pharmacological agents that inhibit one or more functions of the integrase. The procedures are not mutually exclusive, that is, more than one of them can be used at a time. For example, both the integrase and the binding sites may be non-functional, or the integrase and the PPT site may be non-functional, or the binding sites and the PPT site may be non-functional, or all of them may be non-functional. functional

As indicated above, one method is to make and use a non-functional integrase. Integrase is involved in the cleavage of viral double-stranded DNA from viral blunt ends and in the binding of the ends to 5'-phosphates in the two strands of a chromosomal target site. Integrase has three functional domains: the N-terminal domain, which contains a zinc binding motif (HHCC), the core of the core domain, which contains the catalytic core and a conserved DD35E motif (D64, D116, E152 in HIV- 1), and a C-terminal domain, which has DNA binding properties. Point mutations introduced into the integrase are sufficient to alter normal function. Many mutations of integrase have been constructed and characterized (see, Philpott and Thrasher, Human Gene Therapy 18: 483, 2007; Apolonia, Thesis presented at University College London, April 2009, p.

82-97; Engelman et al. J Virol 69: 2729, 1995; Nightingale et al. Mol Therapy, 13: 1121, 2006). The sequence encoding the integrase protein can be deleted or mutated to render the protein inactive, preferably without significantly impairing reverse transcriptase activity or nuclear targeting, thus preventing only the integration of the provirus into the genome of the target cell. Acceptable mutations can reduce integrase catalysis, chain transfer, att site binding, host chromosomal DNA binding and other functions. For example, a single substitution of aspartic acid to asparagine in the rest of the HIV integrase or SIV completely abolishes the integration of the viral DNA. Integrase deletions will generally be limited to the C-terminal domain. Removal of the coding sequence for residues 235-288 results in a useful non-functional integrase (Engelman et al., J Virol 69: 2729, 1995). As additional examples, mutations can be generated, for example, Asp64 (the numbers of residues are given for HIV-1, the numbers of corresponding residues for the integrase of other lentiviruses or retroviruses can be easily determined by one skilled in the art) ( eg, D64E, D64V), Asp116 (eg, D116N), Asn120 (eg, N120K), Glu152, Gln148 (eg, Q148A), Lys156, Lys159, Trp235 (p. eg, W235E), Lys264 (eg, K264R), Lys266 (eg, K266R), Lys273 (eg, K273R). Other mutations can be constructed and tested for integration, transgenic expression and any other desirable parameter. The assays for these functions are well known. Mutations can be generated by any of a variety of techniques, including site-directed mutagenesis and chemical synthesis of the nucleic acid sequence. A mutation can be made or more than one of these mutations can be present in the integrase. For example, an integrase can have mutations in two amino acids, three amino acids, four amino acids, etc.

Alternatively or in combination with the use of integrase mutant (s), the binding sites ( att ) can also be mutated in U3 and U5. The integrase binds to these sites and the 3'-terminal dinucleotide is cleaved at both ends of the vector genome. A CA dinucleotide is found at the 3 'end processed; CA is necessary for processing, mutation of nucleotides blocks integration into the chromosome of the host. The A of the CA dinucleotide is the most critical nucleotide for integration, and mutations at both ends of the genome will give the best results (Brown et al., J Virol 73: 9011 (1999).) In an illustration, the CA at each end changes to TG In other illustrations, the CA at each end is changed to TG at one end and GT at the other end.In other illustrations, the CA is removed at each end, in other illustrations, the A from CA is deleted at each end.

The integration can also be inhibited by mutation or elimination of the polypurine tract (PPT) (WO 2009/076524), located in the vicinity of the 3 'LTR. PPT is a polyurine sequence of about 15 nucleotides that can serve as a primer binding site for the synthesis of positive strand DNA. In this case, mutations or eliminations of PPT are directed to the process of reverse transcription. Without wishing to be subject to a mechanism, when mutating or eliminating PPT, the production of linear DNA is radically reduced and, essentially, only 1-LTR DNA circles are produced. The integration requires a linear double-stranded DNA vector genome, and integration is essentially eliminated without it. As indicated above, a PPT can be made non-functional by mutation or by elimination. Typically, the PPT of about 15 nt integer is eliminated, although in some embodiments, shorter deletions of 14 nt, 13, nt, 12 nt, 11 nt, 10 nt, 9 nt, 8 nt, 7 nt, 6 can be made. nt, 5 nt, 4 nt, 3 nt and 2 nt. When mutations are made, multiple mutations are typically made, especially in the 5 'half of the PPT (McWilliams et al., J Virol 77: 11150, 2003), although single and double mutations in the first four bases still reduce the transcription. Mutations made at the 3 'end of PPT generally have a more remarkable effect (Powell and Levin J Virol 70: 5288, 1996).

These different methods for making a non-integrating vector genome can be used individually or in combination. The use of more than one procedure can be used to construct a fail-safe vector by means of redundant mechanisms. Therefore, mutations or eliminations of PPT can be combined with mutations or deletions of the att site or with integrase mutations or mutations or eliminations of PPT can be combined with mutations or deletions of att sites as well as with integrase mutations. Similarly, AT site mutations or deletions and integrase mutations can be combined with each other or with mutations or deletions of PPT.

A further method for improving the safety of the pseudotyped lentiviral particles involves modifying the plasmid comprising the gag / pol genes to eliminate sites of potential recombination with the lentiviral vector. This method can be used individually or in combination with any of the methods described herein. For example, the gag and pol genes can be of "optimized codons" of mammalian or human, that is, at least 50%, 60%, 70%, 75%, 80%, 85%, 90% of 95% of the codons of the gag and / or pol genes are replaced by codons encoding the same amino acid but which are preferred by mammalian cells, e.g. eg, human cells, thereby improving or optimizing expression in mammalian cells, e.g. eg, human. For codon optimization of the gag and pol genes, a polynucleotide is generated that alters the "natural type" codons to codons most frequently used in the human genome. However, in HIV, certain parts of the genome must substantially retain the original codons (eg, at least 75%, 80%, 85%, 90%, 95% or 100% of the original codons) in order to to allow the displacement of the reading frame that is required to synthesize Gag and Pol starting from the same start codon of its mRNA. The translation of Gag-Pol requires a change of the reading frame in the 5 'direction (change -1) at the p7 / p1 junction, around codon 432 of the mRNA. The Gag-Pol translation then continues in this new reading frame until a stop codon is reached approximately 3,000 nucleotides later. The displacement of the ribosomal reading frame -1 is a highly controlled event that requires both a specific sliding consensus sequence and a secondary RNA structure in the 3 'direction that causes the ribosome to stop. Thus, according to the present disclosure, the nucleic acid encoding gag and pol contains a "non-optimized window": a region that starts around position 1228 (or starts around position 1218-1298, or 1218-1238, or 1228-1238, or 1218-1228, or 1228-1298, or 1238-1248, or 1248-1258, or 1258-1268, or 1268-1278, or 1278-1288, or 1288-1298) of SEQ ID NO : 54 (the gag-pol open reading frame) and ends at position 1509 (or ends around position 1373-1558, or 1373-1509, or 1373-1500, or 1373-1470, or 1373-1440, or 1373-1410, or 1373-1401, or 1373 1392, or 1373-1383, or 1401-1509, or 1440-1509, or 1499-1519, or 1499-1509, or 1509-1519) of SEQ ID NO: 54 which has not been substantially modified with respect to the wild-type gag-pol sequence. In other words, the non-optimized window substantially retains the original codons (eg, at least 90%, 95% or 100% of the original codons). It is understood that the non-optimized window corresponding to nucleotides 1228-1509 of SEQ ID NO: 54 may have a different actual nucleotide numbering when SEQ ID NO: 54 is mutated or is part of a larger nucleotide sequence; for example, if a plasmid contains 100 nucleotides 5 'of SEQ ID NO: 54, then the non-optimized window will correspond to nucleotides 1328-1609 in the plasmid. In some embodiments, the region of nucleotides 1229 through 1298 retains substantially the original codons, and / or the region of nucleotides 1510-1558 is codon-optimized. In some embodiments, the codon-optimized polynucleotide encoding Gag and Pol comprises SEQ ID NO: 54. In some embodiments, the codon-optimized codon gag-pol polynucleotide comprises the sequence of SEQ ID NO: 55.

In some or any of the embodiments, the plasmid encoding the human codon optimized gag and pol genes (and comprising the non-optimized window described above) also lacks a Rev response element (RRE). The removal of the RRE also eliminates another region of homology and the potential recombination between the gag / pol plasmid and the lentiviral vector plasmid. In combination, said method eliminates or minimizes regions of homology between the gag / pol plasmid and the lentiviral vector, thus minimizing the possibilities of recombination between these plasmids that could result in a virus competent for replication. It is understood that the RRE can still be present in the cell and the packaging process, but is present in a different plasmid.

Regulatory elements

As described herein, the genome of the viral vector comprises a sequence of interest that is convenient to express in target cells. Typically, the sequence of interest is located between the LTR 5 'and LTR 3' sequences. In addition, the sequence of interest preferably is in a functional relationship with other genetic elements, for example, transcriptional regulatory sequences that include promoters or enhancers, to regulate the expression of the sequence of interest in a particular manner. In certain cases, the useful transcriptional regulatory sequences are those that are highly regulated with respect to activity, both temporally and spatially. Expression control elements that can be used to regulate the expression of the components are known in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, enhancers and other regulatory elements.

The sequence of interest and any other expressible sequence are typically in a functional relationship with the regulatory sequences of the internal promoter / enhancer. An "internal" promoter / enhancer is one that is located between the 5 'LTR and 3' LTR sequences in the viral vector construct and is operably linked to the sequence of interest. The internal promoter / enhancer can be any promoter, enhancer or promoter / enhancer combination that is known to increase the expression of a gene with which it is in a functional relationship. A "functional relationship" and "operably linked" means, without limitation, that the sequence is in the correct location and orientation with respect to the promoter and / or enhancer, so that the sequence of interest will be expressed when the promoter and / or enhancer get in contact with the right molecules.

The choice of an internal promoter / enhancer is based on the desired expression pattern of the sequence of interest and the specific properties of the known promoters / enhancers. Therefore, the internal promoter can be constitutively active. Non-limiting examples of constitutive promoters that can be used include the ubiquitin promoter (U.S. Patent No. 5,510,474, WO 98/32869), CMV (Thomsen et al., PNAS 81: 659, 1984; from U.S. Patent No. 5,168,062), beta-actin (Gunning et al., 1989 Proc. Natl. Acad. Sci. USA 84: 4831-4835) and pgk (see, for example, Adra et al., 1987 Gene 60: 65-74; Singer-Sam et al., 1984 Gene 32: 409-417; and Dobson et al., 1982 Nucleic Acids Res. 10: 2635-2637). In some embodiments, the promoter used to control the expression of the antigens encoded by the genome of the pseudotyped lentiviral vector is an introns deficient promoter. In some embodiments, the human ubiquitin-C promoter (UbiC) is used to control the expression of the antigens encoded by the genome of the pseudotyped lentiviral vector. In different embodiments, the UbiC promoter has been modified to remove introns, i.e., the promoter is deficient in introns. The full-length UbiC promoter is 1250 nucleotides. The intron starts at 412 and goes to the end (412 1250). This region can be eliminated in order to minimize heterogeneous viral genomic transcripts. The viral genome of HIV has a natural intron within it. Therefore, a lentivirus comprising a UbiC promoter would have a total of 2 introns in the lentivirus genome. The UbiC intron can exist in spliced and non-spliced forms. The elimination of UbiC introns eliminates the possibility of heterogeneous viral transcripts and ensures homogeneity in the pseudotyped lentiviral particles delivered.

Alternatively, the promoter can be a tissue-specific promoter. In some preferred embodiments, the promoter is a target cell-specific promoter. For example, the promoter can be any product expressed by dendritic cells, including CD11c, CD103, TLR, DC-SIGN, BDCA-3, DEC-205, dC | R2, mannose receptor, Dectin-1, Clec9A, MHC class II. In addition, promoters can be selected to allow inducible expression of the sequence of interest. A number of systems for inducible expression are known in the art, including the tetracycline responsive system, the lac operator-repressor system, as well as promoters that respond to a variety of environmental or physiological changes, including thermal shock, metal ions, such as promoter of metallothionein, interferons, hypoxia, steroids, such as progesterone or glucocorticoid receptor promoter, radiation, such as VEGF promoter. A combination of promoters may also be used to obtain the desired expression of the gene of interest. The person skilled in the art will be able to select a promoter based on the desired expression pattern of the gene in the organism or target cell of interest.

The viral genome may comprise at least one promoter responsive to RNA polymerase II or III. This promoter may be operably linked to the sequence of interest and may also be linked to a terminator sequence. In addition, more than one RNA polymerase II or III promoter can be incorporated. The promoters of RNA polymerase II and III are well known to those skilled in the art. A suitable variety of RNA polymerase III promoters can be found, for example, in Paule and White, Nucleic Acids Research., Vol. 28, p. 1283-1298 (2000). The promoters of RNA polymerase II or III also include any synthetic or genetically modified DNA fragment that can direct RNA polymerase II or III to transcribe RNA coding sequences in the 3 'direction. In addition, the promoter or promoters of RNA polymerase II or III (Pol II or III) used as part of the genome of the viral vector may be inducible. Any suitable Pol II or III inducible promoter can be used with the methods of the disclosure. Particularly suitable Pol II or III promoters include the tetracycline sensitive promoters provided in Ohkawa and Taira, Human Gene Therapy, vol. 11, p. 577 585 (2000) and in Meissner et al. Nucleic Acids Research, vol. 29, p. 1672-1682 (2001).

An internal enhancer in the viral construct may also be present to increase the expression of the gene of interest. For example, the CMV enhancer can be used (Boshart et al., Cell, 41: 521, 1985). Many enhancers have been identified and characterized in viral genomes, such as HIV, CMV and in mammalian genomes (see, GenBank). An enhancer may be used in combination with a heterologous promoter. One skilled in the art will be able to select the appropriate enhancer based on the desired expression pattern.

A viral vector genome will normally contain a promoter that is recognized by the target cell and that is operably linked to the sequence of interest, viral components and other sequences described herein. A promoter is an expression control element formed by a nucleic acid sequence that allows the binding of the RNA polymerase and that transcription occurs. The promoters can be inducible, constitutive, temporarily active or tissue-specific. The activity of inducible promoters is induced by the presence or absence of biotic or abiotic factors. Inducible promoters can be a useful tool in genetic engineering because the expression of the genes to which they are operatively linked can be activated or deactivated at certain stages of the development of an organism, its manufacture or in a particular tissue. The inducible promoters can be grouped as chemically regulated promoters and physically regulated promoters. Typical chemically-regulated promoters include, but are not limited to, alcohol-regulated promoters (eg, promoter of the alcohol dehydrogenase I gene (alcA)), regulated portetracycline promoters (eg, tetracycline-sensitive promoter), steroid-regulated promoter (eg, rat glucocorticoid receptor (GR) -based promoter, human estrogen receptor (ER) -based promoter, ecdysone moth receptor-based promoter and promoters based on the receptor superfamily of steroids / retinoids / thyroids), metal-regulated promoters (eg, promoters based on the metallothionein gene), and promoters related to pathogenesis (eg, promoters based on pathogen-related proteins (PR) of the corn and Arabidopsis). Typical physically regulated promoters include, but are not limited to, temperature regulated promoters (eg, heat shock promoters), and light regulated promoters (eg, SSU soy promoters).

One skilled in the art will be able to select a suitable promoter based on the specific circumstances. Many different promoters are well known in the art, as are methods for operatively linking the promoter to the gene to be expressed. Both natural promoter sequences and many heterologous promoters can be used to direct expression in the packaging cell and the target cell. However, heterologous promoters are preferred, since they generally allow greater transcription and higher yields of the desired protein compared to the natural promoter.

The promoter can be obtained, for example, from genomes of viruses such as polyoma virus, fowlpox virus, adenovirus, bovine papillomavirus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis B virus and simian virus 40 (SV40). . The promoter can also be, for example, a heterologous mammalian promoter, e.g. e.g., the actin promoter or an immunoglobulin promoter, a heat shock promoter, or the promoter normally associated with the natural sequence, provided that said promoters are compatible with the target cell. In one embodiment, the promoter is the viral promoter that is naturally found in a viral expression system. In some embodiments, the promoter is a specific promoter of dendritic cells. The specific promoter of dendritic cells can be, for example, the CD11c promoter.

Transcription can be increased by inserting an enhancer sequence into the vector (s). Enhancers are typically cis-acting DNA elements, typically about 10 to 300 bp in length, which act on a promoter to increase its transcription. Many sequences of mammalian gene enhancers (globin, elastase, albumin, alpha-fetoprotein and insulin) and of eukaryotic cell viruses are now known. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer can be spliced into the vector 5 'or 3' of the antigen-specific polynucleotide sequence, but preferably located at a 5 'site of the promoter.

Expression vectors may also contain sequences necessary for the termination of transcription and to stabilize the mRNA. These sequences are often found in the 5 'and occasionally 3' untranslated regions of eukaryotic or viral DNA or cDNA and are well known in the art.

The genome of the viral vector may also contain additional genetic elements. The types of elements that can be included in the construction are not limited in any way and can be chosen to achieve a particular result. For example, a signal that facilitates the nuclear entry of the viral genome into the target cell can be included. An example of such a signal is the flap signal of HIV-1. In addition, elements that facilitate the characterization of the site of integration of provirus in the target cell can be included. For example, a sequence of amber tRNA suppressor may be included in the construct. An insulating sequence, e.g. eg, chicken p-globin in the construction of the viral genome. This element reduces the possibility of silencing a provirus integrated in the target cell due to the effects of methylation and heterochromatinization. In addition, the insulator can protect the internal enhancer, promoter and exogenous gene from the positive or negative positional effects of the surrounding DNA at the site of integration into the chromosome. In addition, the vector genome may contain one or more genetic elements designed to improve the expression of the gene of interest. For example, an element sensitive to groundhog hepatitis virus (WRE) can be placed in the construction (Zufferey et al., 1999. J. Virol. 74: 3668 3681; Deglon et al., 2000. Hum. Gene Ther. 11: 179-190).

The genome of the viral vector is typically constructed in a plasmid form that can be transfected into a producer or packaging cell line. The plasmid generally comprises sequences useful for the replication of the plasmid in bacteria. Such plasmids are well known in the art. In addition, the vectors that include a prokaryotic origin of replication may also include a gene whose expression confers a detectable or selectable marker, such as resistance to a drug. The products of resistance to typical bacterial drugs are those that confer resistance to ampicillin or tetracycline.

Plasmids containing one or more of the components described herein are easily constructed using conventional techniques well known in the art. For the analysis to confirm the correct sequences in constructed plasmids, the plasmid can be replicated in E. Coli, purified and analyzed by digestion with restriction endonucleases or determine its DNA sequence by conventional methods.

Constructed vectors for transient expression in mammalian cells can also be used. Transient expression involves the use of an expression vector that can be replicated efficiently in a host cell, such that the host cell accumulates many copies of the expression vector and, in turn, synthesizes high levels of a polypeptide encoded by the antigen-specific polynucleotide in the expression vector. See, Sambrook et al., See above, p. 16.17-16.22. Other vectors and methods suitable for adaptation to the expression of polypeptides are well known in the art and readily adapt to specific circumstances.

Using the teachings provided herein, one skilled in the art will recognize that the efficacy of a particular expression system can be assayed by transfecting packaging cells with a vector comprising a gene encoding an indicator protein and measuring expression using a technique suitable, for example, by measuring the fluorescence of a green fluorescent protein conjugate. Suitable reporter genes are well known in the art.

Types of sequences of interest

The sequence of interest is not limited in any way and includes any nucleic acid that one skilled in the art wishes to have integrated, transcribed and expressed in the target cell. The product can be a protein or a nucleic acid. The sequence of interest can encode a protein or a nucleic acid molecule, including siRNA, microRNA, a self-complementary double-stranded RNA in which the complementary region has a length greater than about 20 ribonucleotides, or an RNA that is complementary to a messenger RNA, where the binding of said complementary RNA (antiparallel) to the messenger RNA blocks its ability to be translated into protein. In some cases, the sequence of interest can encode an antigen against which an immune response is desired. In particular, tumor antigens and antigens of infectious diseases of agents such as HIV, HSV, HCV, HPV, malaria or tuberculosis are desirable. In addition, multiple sequences of interest can be included in a single vector.

In certain cases, the sequence of interest may be a gene encoding a small inhibitory RNA (siRNA) or a microRNA (miRNA) of interest that down-regulates the expression of a molecule. For example, the gene encoding a siRNA or a microRNA can be used to down regulate the expression of negative regulators in a cell, including those that inhibit the activation or maturation of dendritic cells. SiRNAs and microRNAs are well known in the art (Fire et al., Nature 391: 806, 1998, see also "The RNA Interference Resource" by Applied Biosystems, Trang et al., Oncogene Suppl 2: S52, 2008; Taganov , K., et al., 2007. Immunity 26: 133-137; Dahlberg, JE and E. Lund, 2007. Sci. STKE 387: Pe25; Tiemann and Rossi, EMBO Mol Med 1: 142, 2009). Alternatively, the sequence of interest may encode a self-complementary double-stranded RNA in which the complementary region is greater than about 20 ribonucleotides in length, or an antiparallel RNA that is greater than about 20 ribonucleotides in length. It will be appreciated by those skilled in the art that antiparallel siRNA, siRNA, dsRNA, and RNA molecules can be expressed from an RNA polymerase III promoter or, alternatively, they can be a component of an antisense RNA that is transcribed from a promoter of RNA polymerase II.

In addition, the sequence of interest can encode more than one product. In some configurations, the sequence to be delivered may comprise multiple genes encoding at least one protein, at least one siRNA, at least one microRNA, at least one dsRNA or at least one antiparallel RNA molecule or any combination thereof. For example, the sequence to be delivered may include one or more genes encoding one or more antigens against which an immune response is desired. The one or more antigens may be associated with a single disease or disorder, or may be associated with multiple diseases and / or disorders. In some cases, a gene encoding an immune regulatory protein may be included together with a gene encoding an antigen against which an immune response is desired, and the combination may elicit and regulate the immune response in the desired direction and magnitude. In other cases, a sequence encoding a siRNA, microRNA, dsRNA or antiparallel RNA molecule can be constructed with a gene encoding an antigen against which an immune response is desired, and the combination can regulate the extent of the immune response. The products can be produced as an initial fusion product in which the coding sequence is in functional relation to a promoter. Alternatively, the products may be encoded separately and each coding sequence in functional relation to a promoter. The promoters can be the same or different.

In certain configurations, the vectors contain polynucleotide sequences that encode maturation / stimulation factors of dendritic cells. Exemplary stimulator molecules include GM-CSF, IL-2, IL-4, IL-6, IL-7, IL-15, IL-21, IL-23, TNFa, B7.1, B7.2, 4- 1BB, ligand CD40 (CD40L), CD40 drug-induced (iCD40), and the like. These polynucleotides are typically under the control of one or more regulatory elements that direct the expression of the coding sequences in dendritic cells. The maturation of the dendritic cells contributes to a satisfactory vaccination (Banchereau, J and Palucka, AK Nat. Rev. Immunol., 5: 296-306 (2005); Schuler, G. et al., Curr. Opin. Immunol., 15: 138 -147 (2003); Figdor, CG et al., Nat. Med. 10: 475-480 (2004)). Ripening can transform DC cells actively involved in antigen capture to specialized cells for priming T cells For example, linking CD40 by CD40L in T helper cell ^c D4 is a critical DC maturation signal, which results in a potent activation of CD8 T cells. These stimulatory molecules are also called maturation factors or factors that stimulate maturation. Immune control sites represent significant barriers to the activation of functional cellular immunity in cancer, and antagonist antibodies specific for inhibitory ligands in T cells, including CTLA4 and programmed death-1 (PD-1), are examples of targeted agents that are being evaluated in the clinics. An important mechanism of tolerance in chronic infections and cancer is the functional depletion of Ag-specific T cells that express high levels of PD-1. Since it has been shown that the potency of therapeutic immunization is significantly improved by the combination with the control of the immune control point, as a non-limiting example, those skilled in the art will appreciate that an alternative method to inhibit the immune control point is to inhibit the expression of the programmed death (PD) ligands one and two (PD-L1 / L2). One way to achieve inhibition is by expression of RNA molecules such as those described herein, which repress the expression of PD-L1 / L2 in DC transduced with the lentivirus vector encoding one or more of the RNA molecules. The maturation of the DCs or the expression of particular elements, such as the immune control points, for example PD-1 ligands, can be characterized by flow cytometric analysis of the regulation by increase of a surface marker such as MHC II and the profile of chemokines and expressed cytokines.

A sequence encoding a detectable product, typically a protein, can be included to allow identification of the cells that are expressing the desired product. For example, a fluorescent marker protein, such as the green fluorescent protein (GFP), is incorporated into the construct together with a sequence of interest (eg, which encodes an antigen). In other cases, the protein may be detectable by an antibody or the protein may be an enzyme that acts on a substrate to produce a detectable product, or a product that allows the selection of a transfected or transduced target cell, for example, confers resistance to drug, as hygromycin resistance. Typical selection genes encode proteins that confer resistance to antibiotics or other toxins suitable for use in eukaryotic cells, e.g. e.g., neomycin, methotrexate, blasticidin, among others known in the art, or complement auxotrophic deficiencies, or provide critical nutrients retained from the media. The selectable marker may optionally be present in a separate plasmid and introduced by cotransfection.

One or more multicistronic expression units can be used that include two or more of the elements (eg, sequence (s) of interest, the shell molecule, DC maturation factors) necessary for the production of the desired virus in packaging cells. The use of multicistronic vectors reduces the total number of nucleic acid molecules required and, therefore, avoids the possible difficulties associated with the coordination of the expression of multiple vector genomes. In a multicistronic vector, the different elements to be expressed are operatively linked to one or more promoters (and other expression control elements, as necessary). In some configurations, a multicistronic vector comprises a sequence of interest, a sequence encoding a reporter product and viral elements. The sequence of interest typically encodes an antigen and, optionally, a DC maturation factor. Sometimes, the multicistronic vector comprises a gene encoding an antigen, a gene that encodes a DC maturation factor and viral elements.

The pseudotyped lentiviral vectors described can be genetically modified to express more than one, e.g. eg, two, three or four, antigens at the same time. Several methods are known in the art to simultaneously express more than one gene from a single vector. For example, vectors may comprise multiple promoters fused to the open reading frames (ORFs) of the genes, insertion of splicing signals between genes, fusion of genes whose expressions are directed by a single promoter, insertion of proteolytic cleavage sites between genes, insertion of internal ribosome entry sites (IRES) between genes, insertion of bidirectional promoters between genes and / or 2A "self-cleaving" peptides. Each component to be expressed in a multicistronic expression vector can be separated, for example, by an internal ribosome entry site element (IRES) or a viral 2A element, to allow the separate expression of the different proteins thereof. promoter. IRES elements and 2A elements are known in the art (U.S. Patent No. 4,937,190; Philip et al., 2004. Traffic 5: 616-626). In one embodiment, oligonucleotides encoding sequences of furin cleavage sites (RAKR) (Fang et al., 2005. Nat. Biotech 23: 584-590) attached to sequences similar to 2A FMD virus are used (FMDV ; F2A), porcine teschovirus-1 (P2A), equine rhinitis virus A (ERAV; E2A) and Thosea assigns virus (TaV; T2A) (Szymczak et al., 2004. Nat. Biotechnol., 22: 589-594 ) to separate the genetic elements in a multicistronic vector. The consensus sequence of peptide 2A is D (V / I) EXNPGP (SEQ ID NO: 56). In some embodiments, the lentiviral vector comprises a polynucleotide encoding T2A (SEQ ID NO: 57). In some embodiments, peptide 2A is encoded by the polynucleotide of s Eq ID NO: 52. The efficacy of a vector Particular multicistronic can be easily assayed by detecting the expression of each of the genes using conventional protocols.

The expression of two or more antigens can also be achieved using the internal ribosome entry sites (IRES). IRES allow eukaryotic ribosomes to enter and scan an mRNA at a position other than the 5 'm7 G-cap structure. If it is located internally, p. 3 'of a first coding region (or cistron), an IRES will allow translation of a second coding region within the same transcript. The second coding region is identified by the first ATG found after the IRES. Exemplary IRES elements include viral IRES, such as the IRES of picornaviruses and the IRES of cardioviruses (see, e.g., U.S. Patent No. 4,937,190) and non-viral IRES elements found in the 5 'UTR (e.g., transcript elements that encode the immunoglobulin heavy chain binding protein (BiP) (Macejak et al., Nature, 35390-4, 1991); Drosophila Antennapedia (Oh et al. , Genes Dev. 6: 1643-53, 1992) and Ultrabithorax (Ye et al., Mol Cell Biol., 17: 1714-21, 1997), fibroblast growth factor 2 (Vagner et al., Mol. Biol., 15: 35-44, 1995), eIF4G initiation factor (Gan et al., J. Biol. Chem. 273: 5006-12, 1998), c-myc protooncogene (Nanbru et al., J. Biol. Chem., 272: 32061-6, 1995; Stoneley, Oncogene, 16: 423-8, 1998); and vascular endothelial growth factor (VEGF) (Stein et al., Mol. Cell Biol., 18: 3112-9). , 1998).

Expression of two or more antigens can also be achieved using bidirectional promoters, i.e., a promoter region or two consecutive cloned promoters whose read addresses move away from each other, and from which two open reading frames are transcribed which flank the promoter region. Examples of such promoters include the PDGF-A promoters, JC neurotrophic virus, BRCA1, transcobalamin II and dipeptidylpeptidase IV.

In a specific example, the genome of the viral vector comprises: a cytomegalovirus enhancer / promoter (CMV) sequence; the R and U5 sequences of the 5 'LTR of HIV; a packing sequence (y); the flap signal of HIV-1; an internal enhancer; an internal promoter; a gene of interest; the element sensitive to the woodchuck hepatitis virus; a sequence of the amber suppressor of tRNA; an element U3 with a deletion of its enhancer sequence; the p-globin chicken insulator; and the R and U5 sequences of the 3 'LTR of HIV. In some examples, the vector genome comprises an intact lentiviral LTR 5 'and a self-activating LTR 3' (Iwakuma et al., Virology 15: 120, 1999).

The construction of the vector genome can be achieved using any suitable genetic engineering technique known in the art, including, without limitation, conventional restriction endonuclease digestion techniques, ligation, transformation, plasmid purification and DNA sequencing, for example. , as described in Sambrook et al. (1989. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY.), Coffin et al. (Retroviruses, Cold Spring Harbor Laboratory Press, NY (1997)) and "RNA Viruses: A Practical Approach" (Alan J. Cann, Ed., Oxford University Press, (2000).

In some embodiments, the sequence of interest encodes at least one antigen. Any antigen that is associated with a dendritic cell disease or disorder can be delivered using the viral particles as described herein. An antigen that is associated with the disease or disorder is identified for the preparation of a viral particle that targets dendritic cells. Antigens associated with many diseases and disorders are well known in the art. It can be known previously that an antigen is associated with the disease or disorder, or can be identified by any method known in the art. For example, an antigen for a type of cancer that a patient is suffering from may be known, such as a tumor-associated antigen or may be identified from the tumor itself by any of a variety of methods known in the art.

Tumor-associated antigens are known for a variety of cancers including, for example, renal cell carcinoma, prostate cancer, melanoma and breast cancer. In some breast cancers, for example, the Her-2 receptor is overexpressed on the surface of cancer cells. Exemplary tumor antigens include, but are not limited to, MAGE, p. eg, MAGE-A3 and MAGE-A1, BAGE, RAGE and NY-ESO-1, which are unmutated antigens expressed in areas with immune privileges of the testes and in a variety of tumor cells; lineage-specific tumor antigens, such as antigen of the melanocyte-melanoma lineage, MART-1 / Melan-A, gp100, gp75, mda-7, tyrosinase and tyrosinase-related protein, p. eg, TRP2; renal cell carcinoma - 5T4, SM22-alpha, carbonic anhydrases I and IX (also known as G250), hypoxia-inducible factors (eg, HIF-1alpha and HIF-2alpha), VEGF or membrane-specific antigen prostate (PSMA), prostate specific antigen (PSA), prostatic acid phosphates and epithelial antigen with six transmembrane domains of the prostate (STEAP), NKX3.1, which are antigens expressed in normal and neoplastic cells derived from the same tissue; proteins / peptides epitopes derived from genes mutated in tumor cells or genes transcribed at different levels in tumors compared to normal cells, such as the enzyme telomerase, survivin, mesothelin, mutated ras, rearrangement bcr / abl, Her2 / neu, p53 mutated or natural type, cytochrome P4501B1, and abnormally expressed intron sequences, such as N-acetylglucosaminyltransferase-V; clonal rearrangements of immunoglobulin genes that generate unique idiotypes in myeloma and B-cell lymphomas; proteins / peptides epitopes derived from oncoviral processes, such as proteins E6 and E7 of the human papillomavirus; non-mutated oncofetal proteins with selective tumor expression, such as carcinoembryonic antigen and alpha-fetoprotein. A series of antigens associated with tumors have been reviewed (see, for example, "Tumor-Antigens Recognized By T-Lymphocytes," Boon T, Cerottini JC, Vandeneynde B, Vanderbruggen P, Vanpel A, Annual Review of Immunology 12: 337-365, 1994; "A listing of human tumor antigens recognized by T cells," Renkvist N, Castelli C, Robbins PF, Parmiani G. Cancer Immunology Immunotherapy 50: (1) 3-15 MAR 2001).

In some or any of the embodiments described herein, the pseudotyped lentiviral vector comprises a polynucleotide that encodes MAGE-A3 (SEQ ID NO: 58). In some embodiments, MAGE-A3 is encoded by the polynucleotide of SEQ ID NO: 50. In some embodiments, MAGE-A3 is encoded by the polynucleotide of at least 40, 50, 60, 70, 80, 90, 95, 99 by percent identity with SEQ ID NO: 50. In some embodiments, the pseudotyped lentiviral vector comprises a polynucleotide that encodes an MAGE-A3 fragment of at least 40, 50, 60, 70, 80, 90, 100, 110, 120 , 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or 310 amino acids of SEQ ID NO: 58. In some embodiments , the polynucleotide encodes a variant of MAGE-A3 having at least 40, 50, 60, 70, 80, 90, 95, 99 percent identity with SEQ ID NO: 58. In some embodiments, the pseudotyped lentiviral vector comprises a polynucleotide encoding NY-ESO-1 (SEQ ID NO: 59). In some embodiments, NY-ESO-1 is encoded by the polynucleotide of SEQ ID NO: 51. In some embodiments, MAGE-A3 is encoded by the polynucleotide of at least 40, 50, 60, 70, 80, 90, 95 percent identity with SEQ ID NO: 51. In some embodiments, the pseudotyped lentiviral vector comprises a polynucleotide that encodes a fragment of NY-ESO-1 of at least 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160 or 170 amino acids of SEQ ID NO: 59. In some embodiments, the polynucleotide encodes a variant of NY-ESO-1 having at least 40, 50, 60, 70, 80 , 90, 95, 99 percent identity with SEQ ID NO: 59. In some embodiments, the pseudotyped lentiviral vector comprises a polynucleotide encoding MAGE-A3 (SEQ ID NO: 58) and a polynucleotide encoding NY-ESO- 1 (SEQ ID NO: 59). In some or any of the embodiments, NY-ESO-1 and MAGE-A3 are expressed from the same transcript as a fusion protein comprising NY-ESO-1 and MAGE-A3 and a peptide A2 of self-cleavage between the two antigens , p. eg, SEQ ID NO: 56 or 57. The antigens may be in any order (eg, NY-ESO-1 first and MAGE-A3 second, or MAGE-A3 first and NY-ESO-1 second).

The antigen may also be an antigen associated with an infectious disease, such as, for example, HIV / AIDS. The antigen can be, for example, gp120 (Klimstra, WB, et al 2003. J Virol 77: 12022-12032; Bernard, KA, et al., 2000. Virology 276: 93-103; Byrnes, AP, et al. 1998. J Virol 72: 7349-7356). Other example antigens include, but are not limited to: gag, pol, env, tat, nef and rev (Lieberman, J. et al., 1997. AIDS Res Hum Retroviruses 13 (5): 383-392; Menendez-Arias, Lethal, 1998. Viral Immunol 11 (4): 167-181).

Examples of viral antigens include, but are not limited to, adenovirus polypeptides, alphavirus polypeptides, calicivirus polypeptides, e.g. eg, a calicivirus capsid antigen, coronavirus polypeptides, distemper virus polypeptides, Ebola virus polypeptides, enterovirus polypeptides, flavivirus polypeptides, hepatitis virus (AE) polypeptides, e.g. eg, a core or surface antigen of hepatitis B, or glycoproteins E1 or E2 of hepatitis C virus, core or non-structural proteins, herpesvirus polypeptides, e.g. eg, a herpes simplex or varicella zoster virus glycoprotein, immunodeficiency virus polypeptides, e.g. eg, the envelope or protease of human immunodeficiency virus, infectious peritonitis virus polypeptides, influenza virus polypeptides, e.g. eg, an influenza A hemagglutinin, neuraminidase or nucleoprotein, leukemic virus polypeptides, Marburg virus polypeptides, orthomyxovirus polypeptides, papillomavirus polypeptides, parainfluenza virus polypeptides, e.g. e.g., hemagglutinin / neuraminidase, paramyxovirus polypeptides, parvovirus polypeptides, pestivirus polypeptides, picornavirus polypeptides, e.g. eg, poliovirus capsid polypeptides, smallpox virus polypeptides, e.g. e.g., a vaccinia virus polypeptide, rabies virus polypeptides, e.g. g., a glycoprotein G of rabies virus, reovirus polypeptides, retrovirus polypeptides and rotavirus polypeptides.

Examples of bacterial antigens include, but are not limited to, Actinomyces polypeptides, Bacillus polypeptides, bacteroid polypeptides, Bordetella polypeptides, Bartonella polypeptides, Borrelia polypeptides, e.g. eg, B. burgdorferi OspA, Brucella polypeptides, Campylobacter polypeptides, Capnocytophaga polypeptides, Chlamydia polypeptides, Clostridium polypeptides, Corynebacterium polypeptides, Coxiella polypeptides, Dermatophilus polypeptides, Enterococcus polypeptides, Ehrlichia polypeptides, Polypeptides of Escherichia, Francisella polypeptides, Fusobacterium polypeptides, Haemobartonella polypeptides, Haemophilus polypeptides, p. Ex., outer membrane protein of H. influenzae type b, Helicobacter polypeptides, Klebsiella polypeptides, L-shaped bacteria polypeptides, Leptospira polypeptides, Listeria polypeptides, Mycobacteria polypeptides, Mycoplasma polypeptides, Neisseria polypeptides, Neorickettsia polypeptides, Nocardia polypeptides, Pasteurella polypeptides, Peptococcus polypeptides, Peptostreptococcus polypeptides, Pneumococcus polypeptides, Proteus polypeptides, Pseudomonas polypeptides, Rickettsia polypeptides, Rochalimaea polypeptides, Salmonella polypeptides, Shigella polypeptides, Polypeptides of Staphylococcus, Streptococcus polypeptides, p. e.g., S. pyogenes M proteins, Treponema polypeptides, and Yersinia polypeptides, e.g. eg, antigens of Y. pestis F1 and V.

Examples of fungal antigens include, but are not limited to, Absidia polypeptides, Acremonium polypeptides, Alternaria polypeptides, Aspergillus polypeptides, Basidiobolus polypeptides, Bipolaris polypeptides, Blastomyces polypeptides, Candida polypeptides, Coccidioides polypeptides, Conidiobolus, Cryptococcus polypeptides, Curvalaria polypeptides, Epidermophyton polypeptides, Exophiala polypeptides, Geotrichum polypeptides, Histoplasma polypeptides, Madurella polypeptides, Malassezia polypeptides, Microsporum polypeptides, Moniliella polypeptides, Mortierella polypeptides, Mucor polypeptides, Paecilomyces polypeptides, Penicillium polypeptides, Phialemonium polypeptides, Phialophora, Prototheca polypeptides, Pseudallescheria polypeptides, Pseudomicrodochium polypeptides, Pythium polypeptides, Rhinosporidium polypeptides, Rhizopus polypeptides, Scolecobasidium polypeptides, Sporothrix polypeptides, Stemphylium polypeptides, Trichophyton polypeptides, Trichosporon polypeptides, and Xylohypha polypeptides .

Examples of protozoan parasite antigens include, but are not limited to Babesia polypeptides, Balantidium polypeptides, Besnoitia polypeptides, Cryptosporidium polypeptides, Eimeria polypeptides, Encephalitozoon polypeptides, Entamoeba polypeptides, Giardia polypeptides, Hammondia polypeptides, Hepatozoon polypeptides, Isospora polypeptides, Leishmania polypeptides, Microsporidia polypeptides, Neospora polypeptides, Nosema polypeptides, Pentatrichomonas polypeptides, Plasmodium polypeptides, e.g. eg, P. falciparum circumsporozoite (PfCSP), sporozoite surface protein 2 (PfSSP2), carboxyl end of liver state antigen 1 (PfLSA1 end c), and exported protein 1 (PfExp-1), Pneumocystis polypeptides , Sarcocystis polypeptides, Schistosoma polypeptides, Theileria polypeptides, Toxoplasma polypeptides, and Trypanosoma polypeptides.

Examples of helminth parasite antigens include, but are not limited to Acanthocheilonema polypeptides, Aelurostrongylus polypeptides, Ancylostoma polypeptides, Angiostrongylus polypeptides, Ascaris polypeptides, Brugia polypeptides, Bunostomum polypeptides, Capillaria polypeptides, Chabertia polypeptides, Cooperia polypeptides, Crenosome polypeptides, Dictyocaulus polypeptides, Dioctophyme polypeptides, Dipetalonema polypeptides, Diphyllobothrium polypeptides, Diplydium polypeptides, Dirofilaria polypeptides, Dracunculus polypeptides, Enterobius polypeptides, Filaroides polypeptides, Haemonchus polypeptides, Polypeptides of Lagochilascaris, Loa polypeptides, Mansonella polypeptides, Muellerius polypeptides, Nanophyetus polypeptides, Necator polypeptides, Nematodirus polypeptides, Oesophagostomum polypeptides, Onchocerca polypeptides, Opisthorc polypeptides his, Ostertagia polypeptides, Parafilaria polypeptides, Paragonimus polypeptides, Parascaris polypeptides, Physaloptera polypeptides, Protostrongylus polypeptides, Setaria polypeptides, Spirocerca polypeptides from Spirometra polypeptides, Stephanofilaria polypeptides, Strongyloides polypeptides, Strongylus polypeptides, polypeptides of Thelazia, Toxascaris polypeptides, Toxocara polypeptides, Trichinella polypeptides, Trichostrongylus polypeptides, Trichuris polypeptides, Uncinaria polypeptides, and Wuchereria polypeptides.

Examples of ectoparasite antigens include, but are not limited to polypeptides (including protective antigens as well as allergens) of fleas; ticks, including hard and soft ticks; flies, such as mosquitoes, mosquitoes, sand flies, black flies, horseflies, horn flies, deer flies, tsetse flies, stable flies, flies that cause myiasis and beatillas; ants; spiders lice; mites and hemiptera, such as bed bugs and killer bugs.

Once an antigen has been identified and selected, a sequence encoding the desired antigen is identified. Preferably, the sequence comprises a cDNA. Following viral infection, the sequence of interest (eg, one encoding the antigen) is expressed by the target dendritic cells. If they are contacted ex vivo, the target dendritic cells are transferred back to the patient, for example, by injection, where they interact with immune cells that are capable of generating an immune response against the desired antigen. In preferred embodiments, the patient is injected with the recombinant virus where it transduces the target dendritic cells in situ. The dendritic cells then express the particular antigen associated with a disease or disorder to be treated, and the patient is able to mount an effective immune response against the disease or disorder.

The genome of the viral vector can contain a polynucleotide sequence that encodes more than one antigen, and upon transduction of a target dendritic cell, generates immune responses against the multitude of antigens delivered to the cell. In some embodiments, the antigens are related to a single disease or disorder. In other embodiments, the antigens are related to multiple diseases or disorders. In some embodiments, the genome of the viral vector contains a polynucleotide sequence encoding MART-1 / Melan-A, NY-ESO-1 and MAGE.

Production of viral particles.

Any of a variety of methods known in the art can be used to produce infectious viral particles, e.g. eg, lentiviral particles, whose genome comprises an RNA copy of the genome of the viral vector. In one method, the genome of the viral vector is introduced into a packaging cell line that contains all the components necessary to package the viral genomic RNA, transcribed from the genome of the viral vector, into viral particles. Alternatively, the genome of the viral vector may comprise one or more genes encoding viral components in addition to one or more sequences of interest. However, in order to prevent replication of the genome in the target cell, the endogenous viral genes required for replication will normally be removed and provided separately in the packaging cell line.

In general, lentiviral vector particles are produced by a cell line that is transfected with one or more plasmid vectors containing the components necessary to generate the particles. These lentiviral vector particles are typically not replication competent, that is, they are capable of only a single cycle of infection. Most of the time, multiple plasmid vectors are used to separate the different genetic components that generate the particles of the lentiviral vector, mainly to reduce the possibility of recombination events that could otherwise generate viruses competent for replication. However, if desired, a single plasmid vector having all the lentiviral components can be used. As an example of a system using multiple plasmid vectors, a cell line is transfected with at least one plasmid that contains the genome of the viral vector (ie, the plasmid of the vector genome), which includes the LTRs, the packaging sequence that acts on cis and the sequence (s) of interest, which are often operably linked to a heterologous promoter, at least one plasmid encoding the viral and structural components of the virus (i.e., the packaging plasmid encoding components) such as, Gag and Pol), and at least one envelope plasmid encoding an Arbovirus envelope glycoprotein. Additional plasmids can be used to improve the production of retrovirus particles, e.g. eg, Rev expression plasmids, as described herein and known in the art. The viral particles exit through the cell membrane and comprise a nucleus that includes a genome that contains the sequence of interest and an envelope glycoprotein of Arbovirus that targets the dendritic cells. When the Arbovirus glycoprotein is the E2 glycoprotein of the Sindbis virus, the glycoprotein is genetically modified so that it has less binding to the heparan sulfate compared to the E2 glycoprotein of the reference strain HR (SEQ ID NO: 18).

The transfection of packaging cells with plasmid vectors of the present disclosure can be carried out by well-known methods, and the method used is not limited in any way. Various nonviral delivery systems are known in the art, including, for example, electroporation, lipid-based delivery systems including liposomes, "naked" Dn delivery and delivery using polycyclodextrin compounds, such as those described in Schatzlein AG . (2001. Non-Viral Vectors in Cancer Gene Therapy: Principies and Progresses, Anticancer Drugs). Methods of treating salts or cationic lipids are typically used, see, for example, Graham et al. (1973. Virol. 52: 456; Wigler et al. (1979. Proc. Natl. Acad. Sci. USA 76: 1373-76) The method of calcium phosphate precipitation is the most frequently used. other methods can also be used to introduce the vector into cells, including microinjection and fusion of bacterial protoplasts.

The packaging cell line provides the components, which include regulatory and viral structural proteins, which are required in trans for the packaging of viral genomic RNA into lentiviral vector particles. The packaging cell line can be any cell line that is capable of expressing lentiviral proteins and producing functional lentiviral vector particles. Some suitable packaging cell lines include 293 (ATCC CCL X), 293T, HeLa (ATCC CCL 2), D17 (ATCC CCL 183), MDCK (ATCC CCL 34), BHK (ATCC CCL-10) and Cf2Th (ATCC CRL) cells. 1430). The packaging cell line can stably express the necessary viral proteins. Said packaging cell line is described, for example, in U.S. Pat. No. 6,218,181. Alternatively, a packaging cell line can be transiently transfected with nucleic acid molecules that encode one or more viral proteins necessary together with the genome of the viral vector. The resulting viral particles are collected and used to infect a target cell. The gene (s) encoding the glycoprotein (s) is normally cloned into an expression vector, such as pcDNA3 (Invitrogen, CA, USA). Expression vectors of eukaryotic cells are well known in the art and are available from a number of commercial sources. The packaging cells, such as 293T cells, are then cotransfected with the genome of the viral vector encoding a sequence of interest (typically encoding an antigen), at least one plasmid encoding the packaging components of the virus and a vector for the expression of the target molecule. The envelope is expressed in the membrane of the packaging cell and is incorporated in the viral vector.

Virus production is measured as described herein and expressed as IU by volume. IU is an infectious unit, or alternatively Transduction Units (TU ); IU and TU can be used interchangeably as a quantitative measure of the titer of a viral vector particle preparation. As described herein, a virus is produced in which the genome can express a product that can be easily measured. A fluorescent protein, the green fluorescent protein, is preferred. The lentiviral vector is typically non-integrating. The virus is then administered to the target cells and the number of target cells expressing GFP is determined, such as by flow cytometry. Then the title is calculated. The titer is preferably as high as possible, but at least 1 x 105 lU / ml, at least 3 x 105 lU / ml, at least 1 x 106 lU / ml, at least 3 x 106 lU / ml, or at least 1 x 107 uU / ml of cellular supernatant fluid (before any concentration). Alternatively, the titer is at least 80%, at least 90%, at least 95%, at least 100% of the titer of the same lentiviral vector pseudotyped in the same cells with VSV-G envelope.

Production of highly mannosylated viral particles

The envelope protein of the Sindbis virus contains four glycans bound to N, two in the E2 protein and two in the E1 protein. Two N-glycans of the virus produced in mammalian cells in the absence of a mannosidase inhibitor I have a structure with high mannose content (one glycan bound to N E2 and one glycan bound to N E1), while the other two have a complex structure. The two N-glycans of complex structure are exposed on the surface of the envelope protein, while the two structure N-glycans with high mannose content are buried within the trimer center of the envelope proteins. Sindbis virus particles with complex N-linked glycans do not bind DC-SIGN as efficiently as particles with less complex, highly-mannosylated glycoproteins.

In the present description, the authors of the invention demonstrate that viral particles produced in mammalian cells in the presence of the mannosidase inhibitor I, kifunensine, unexpectedly exhibit a significant increase in the binding of DC-SIGN compared to particles produced in the presence of of DMNJ.

In some or any of the embodiments, a virus packaging cell is cultured in the presence of a mannosidase I inhibitor. In some or any of the embodiments, the mannosidase I inhibitor is kifunensine. In some embodiments, kifunensine is present in the medium at a concentration of about 0.01 pg / ml to about 1 mg / ml, from about 0.1 pg / ml to about 10 | jg / ml, of about 0.1 pg / ml to about 9 pg / ml, from about 0.1 pg / ml to about 8 pg / ml, from about 0.1 pg / ml to about 7 pg / ml, from about 0.1 pg / ml to about 6 pg / ml, from about 0.1 pg / ml to about 5 pg / ml, from about 0.1 pg / ml to about 4 pg / ml, from about 0.1 pg / ml to about 3 pg / ml, from about 0.1 pg / ml to about 2 pg / ml, from about 0.1 pg / ml to about 1 pg / ml, from about 0.25 pg / ml to about 10 pg / ml, of about 0.25 pg / ml to about 9 pg / ml, from about 0.25 pg / ml to about 8 pg / ml, from about 0.25 pg / ml to about 7 pg / ml, from about 0.25 pg / ml to about 6 pg / ml, of apr approximately 0.25 pg / ml to about 5 pg / ml, from about 0.25 pg / ml to about 4 pg / ml, from about 0.25 pg / ml to about 3 pg / ml, of about 0.25 pg / ml at about 2 pg / ml or about 0.25 pg / ml at about 1 pg / ml.

In some or any of the embodiments wherein a pseudotyped lentiviral vector particle comprises an E2 glycoprotein of the Sindibis virus and a Vpx protein, the lentiviral particles are produced in the presence of a mannosidase I inhibitor. In some embodiments, the mannosidase inhibitor is deoxanojojirimicina (DMNJ). In preferred embodiments, the mannosidase inhibitor is kifunensine. In some embodiments, JNMD is present in the medium at a concentration of about 1.0 pg / ml to about 1.0 mg / ml, from about 1.0 pg / ml to about 900 pg / ml, of about 1.0 pg / ml to about 800 pg / ml, from about 1.0 pg / ml to about 700 pg / ml, from about 1.0 pg / ml to about 600 pg / ml, from about 1.0 pg / ml to about 500 pg / ml, from about 1.0 pg / ml to about 400 pg / ml, from about 1.0 pg / ml to about 300 pg / ml, from about 1.0 pg / ml to about 200 pg / ml, from about 1.0 pg / ml to about 100 pg / ml, from about 50 pg / ml to about 500 pg / ml, from about 50 pg / ml to about 400 pg / ml, from about 50 pg / ml to about 300 pg / ml, from about 50 pg / ml to about 200 pg / ml, from about 50 pg / ml to about 100 pg / ml, from about 100 pg / ml to about 500 pg / ml , from about 100 pg / ml to about 400 pg / ml, from about 100 pg / ml to about 300 pg / ml, from about 100 pg / ml to about 200 pg / ml, from about 200 pg / ml to about 500 pg. / ml, or from approximately 200 pg / ml to approximately 400 pg / ml.

In some or any of the embodiments, a pseudotyped lentiviral vector particle produced in the presence of a mannosidase I inhibitor (eg, kifunensine) comprises a coat glycoprotein (eg, Sindibis E2 virus), wherein at least 60% of the glycans linked to N comprise a structure of Hands (Mans), Man6, Man ⁷ , Mana and / or Mang. In some embodiments, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at minus 98%, at least 99% or 100% of the glycans bound to N comprise a structure Man ⁵ , Man6, Man ⁷ , Mane and / or Man ⁹ +.

In one scenario, one or more vectors are used to introduce polynucleotide sequences into a packaging cell line for the preparation of a lentiviral vector particle pseudotyped with a Sindbis virus envelope glycoprotein such as E2, as described herein. . In some embodiments, the lentiviral vector particle is highly mannosylated. In some embodiments, the lentiviral vector particle also comprises a Vpx protein or a variant thereof. In still other embodiments, the lentiviral vector particle is highly mannosylated and comprises a Vpx protein or a variant thereof. The vectors may contain polynucleotide sequences encoding the different components of the virus that include the Sindbis virus envelope, a sequence or sequences of interest (which typically encode an antigen), and any components necessary for the production of the virus that is not provided by the cell. of packaging.

The glycosylation profile of a viral envelope protein can be determined by any method known in the art. For example, gel exchange assays of viral glycoproteins treated with glucosidases (eg, EndoH or PNGasaF) or left untreated can be compared. Other methods include cleaving glycans from the viral glycoproteins and separating and identifying the components by HPLC and mass spectrometry methods.

Supply of viruses

The virus can be delivered to a target cell in any manner that allows the virus to contact the target cell, e.g. e.g., a dendritic cell (DC), in which the delivery of a polynucleotide of interest is desired. Sometimes, an adequate amount of virus will be introduced into a human being or other animal directly (in vivo), p. eg, by injection into the body. Suitable animals include, without limitation, horses, dogs, cats, cows, pigs, sheep, rabbits, chickens or other birds. The viral particles can be injected by a series of routes, such as intravenous, intradermal, subcutaneous, intranodal, intraperitoneal or mucosal cavity. The virus can be delivered using a subdermal injection device, such as the devices described in US Pat. UU

7,241,275, 7,115,108, 7,108,679, 7,083,599, 7,083,592, 7,047,070, 6,971,999, 6,808,506, 6,780,171, 6,776,776, 6,689,118, 6,670,349, 6,569. 143, 6,494,865, 5,997,501, 5,848,991, 5,328,483, 5,279,552, 4,886,499. Other injection sites are also suitable, such as directly in organs comprising target cells. For example, intraganglionic injection, intra-venous injection, or intra-medullary bone injection can be used to deliver the virus to the lymph node, spleen, and bone marrow, respectively. Depending on the particular circumstances and the nature of the target cells, the introduction can be accomplished by other means including, for example, inhalation or direct contact with epithelial tissues, for example, those of the eyes, mouth or skin.

Alternatively, target cells are provided and contacted with the virus in vitro, such as in culture dishes. Target cells are typically populations of cells comprising dendritic cells obtained from a healthy subject or a subject in need of treatment or in which it is desired to stimulate an immune response to an antigen. Methods for obtaining cells from a subject are well known in the art and include phlebotomy, surgical excision and biopsy. Human DCs can also be generated by obtaining CD34a + human hematopoietic progenitors and using an in vitro culture method as described elsewhere (eg, Banchereau et al., Cell 106, 271-274 (2001)).

The virus can be suspended in medium and added to the wells of a culture dish, tube or other container. The media containing the virus can be added before plating the cells or after plating the cells. The cells are typically incubated in an appropriate amount of medium to provide viability and to allow adequate concentrations of virus in the medium so that transduction of the host cell occurs. The cells are preferably incubated with the virus for a sufficient time to allow the virus to infect the cells. Preferably, the cells are incubated with virus for at least 1 hour, at least 5 hours or at least 10 hours.

Both in the in vivo and in vitro delivery, an aliquot of viral particles containing a sufficient number can be used to infect the desired target cells. When the target cell is to be cultured, the concentration of the viral particles is generally at least 1 lU / jl, more preferably at least 10 lU / jl, even more preferably at least 300 lU / jl, even more preferably at least 1X104 lU / jl, even more preferably at least 1X105 lU / jl, even more preferably at least 1X106 lU / jl, or even more preferably at least 1X107 lU / jl.

After infection with the virus in vitro, the target cells can be introduced (or reintroduced) into a human or other animal. The cells can be introduced into the dermis, under the dermis or into the peripheral bloodstream. The cells introduced into an animal are preferably cells obtained from that animal, to avoid an adverse immune response. Cells obtained from a donor having a similar immune context can also be used. Other cells that may also be used include those designed to avoid an adverse immune response.

The integration, transcription and / or expression of the sequence or gene (s) of interest, the number of copies of the integrated gene and the location of the integration, for example, can be analyzed in the target cells. Said analysis can be carried out at any time and can be carried out by any method known in the art.

In the subjects to whom a virus is administered, or dendritic cells infected by the virus, the location of the infected cells, expression of the polynucleotide or gene of interest supplied by the virus, stimulation of an immune response, and control of the associated symptoms. with a disease or disorder by any method known in the art.

The methods for infecting cells described above do not depend on individual characteristics specific to the cells. As a result, they easily spread to a variety of animal species. In some cases, the viral particles are delivered to a human or human dendritic cells, and in other cases they are delivered to an animal such as a mouse, horse, dog, cat or mouse or birds. As described herein, the genome of the viral vector is pseudotyped to confer a wide variety of hosts, as well as specificity of the target cell. One skilled in the art would also know the suitable internal promoters and other elements to achieve the desired expression of a sequence of interest in a particular animal species. Therefore, one skilled in the art will be able to modify the method of infecting dendritic cells of any species. Therapeutic and prophylactic immunizations.

Dendritic cells can be infected with a lentivirus vector particle as described herein for the prevention or treatment of a disease or disorder, in particular those for which it would be The activation of an immune response in a patient is beneficial. Many of these diseases are well known. For example, diseases or disorders that are amenable to treatment or prevention by the methods of the present disclosure include, without limitation, cancers, autoimmune diseases and infections, including viral, bacterial, fungal and parasitic infections. In one method, a disease is treated with viral particles (e.g., highly mannosylated viral particles comprising a Vpx protein) described herein in order to deliver a sequence of interest to the dendritic cells, wherein the expression of the sequence of interest produces a specific antigen of the disease and leads to the stimulation of antigen-specific cellular immune responses and humoral immune responses. In general, the sequence of interest encodes an antigen against which an immune response is desired, but is not normally expressed in a dendritic cell. The antigen is expressed and presented by the dendritic cell. The genome of the viral vector can also encode a DC maturation factor.

In a typical use, the viral particles deliver to the dendritic cells sequences encoding an antigen against which an immune response is desired. The delivery can be achieved by contacting dendritic cells with the virus in vitro, after which infected dendritic cells are provided to a patient. Other times, delivery may be achieved by supplying the virus to a subject to infect dendritic cells in vivo. The dendritic cells then stimulate T cells or antigen-specific B cells in a patient to induce humoral and cellular immune responses against expressed antigen. In this way, a patient suffering from a disease or disorder is treated by generating immune cells with a desired specificity.

In some of the viruses, the maturational factors of the DCs that activate and / or stimulate the maturation of the DCs are supplied together with the sequence of interest. Alternatively, DCs are activated by the delivery of DC maturation factors before, simultaneously or after virus delivery. DC maturation factors can be provided separately from the administration of the virus.

As described herein, one or more factors of immune modulation or DC maturation can be encoded by one or more sequences that are contained in the viral genome and are expressed after the virus infects a dendritic cell. Sequences encoding immune modulating factors can also be provided in a separate vector that is cotransfected with the viral vector encoding one or more antigens in a packaging cell line.

The methods described herein can be used for adoptive immunotherapy in a patient. As described above, an antigen against which an immune response is desired is identified. A polynucleotide encoding the desired antigen is obtained and packaged in a recombinant virus. Target dendritic cells are obtained from the patient and transduced with a recombinant virus containing a polynucleotide encoding the desired antigen. The dendritic cells are transferred back to the patient.

The viral particles can be injected in vivo, where they infect DC and provide a sequence of interest, which typically encodes an antigen. The amount of viral particles is at least 3X106 IU, and can be at least 1X107 IU, at least 3X107 IU, at least 1X108 IU, at least 3X108 IU, at least 1X109 IU, or at least 3X109 IU. At selected intervals, the DCs of the lymphoid organs of the receptor can be used to measure expression, for example, by observing the expression of markers, such as GFP or luciferase. Nucleic acid control techniques and measurements of reverse transcriptase (RT) activity can also be used to analyze the biodistribution of viral particles. T cells of peripheral blood mononuclear cells, lymph nodes, spleens, or malignant tissue or infected by target pathogens of receptors treated with lentiviral vector particles can be measured from the magnitude and durability of the response to antigen stimulation. It can be analyzed in tissue cells other than DC, such as epithelial cells and lymphoid cells, the specificity of gene delivery in vivo.

Vaccines often include an adjuvant. The lentiviral vector particles described herein may also be administered together with an adjuvant. The adjuvant can be administered with the recombinant virus particles, before the recombinant virus particles, or after the recombinant virus particles. If administered with the viral particles, the desirable adjuvants do not significantly alter the integrity of the viral particle, such as by altering the viral membrane containing the envelope glycoproteins.

A variety of adjuvants can be used in combination with the virus to elicit an immune response against the antigen encoded in the genome of the viral vector. Preferred adjuvants increase the intrinsic response to an antigen without causing conformational changes in the antigen that affect the qualitative form of the response. Preferred adjuvants include alum, monophosphoryl lipid A (MPL) 3-De-O-acylated (see GB 2220211). QS21 is a triterpene glycoside or saponin isolated from the bark of the Quillaja Saponaria Molina tree found in South America (see Kensil et al., In Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell and Newman, Plenum Press, NY, 1995); U.S. Patent No. 5,057,540). Other adjuvants are oil-in-water emulsions (such as squalene or arachis oil), optionally in combination with immune stimulators, such as monophosphoryl lipid A (see Stoute et al., N. Engl. J. Med. 336, 86-91. (1997)). Another adjuvant is CpG (Bioworld Today, November 15, 1998). Adjuvants can be administered as a component of a therapeutic composition with an active agent or can be administered separately, before, simultaneously or after administration of the therapeutic agent.

A class of adjuvants are aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate. Such adjuvants can be used with or without other specific immunostimulatory agents, such as MPL or 3-DMP, QS21, polymeric or monomeric amino acids such as poly (glutamic acid) or polylysine. Another class of adjuvants are oil-in-water emulsion formulations. Such adjuvants can be used with or without other specific immunostimulatory agents, such as muramyl peptides (eg, N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl normuramyl-L-alanil -D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (MTP-PE) ), N-acetylglucosaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxy-propylamide (DTP-DPP) Theramide.TM.), Or other components of the bacterial cell wall. Oil-in-water emulsions include (a) MF59 (WO 90/14837), which contains 5% squalene, 0.5% Tween 80 and 0.5% Span 85 (optionally containing different amounts of MTP-PE) ) formulated into submicron particles using a microfluidizer such as Microfluidizer Model 110Y (Microfluidics, Newton Mass.), (b) SAF, containing 10% squalane, 0.4% Tween 80, 5% polymer blocked with Pluronic L121 and thr-MDP, either microfluidized in a submicron emulsion or vortexed to generate an emulsion of larger particle size, and (c) Ribi adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2 % of squalene, 0.2% of Tween 80 and one or more components of the bacterial cell wall of the group consisting of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM) and cell wall skeleton (CWS), preferably MPL + CWS (Detox ™). Another class of preferred adjuvants are the saponin adjuvants, such as Stimulon ™. (QS21, Aquila, Worcester, Mass.) Or particles generated there such as ISCOM (immunostimulatory complexes) and ISCOMATRIX. Other adjuvants include complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA). Other adjuvants include cytokines, such as interleukins (IL-1, IL-2 and IL-12), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF).

Another adjuvant that can be used with the compositions herein is identified by the chemical formula (I):

wherein the residues A1 and A2 are independently selected from the group of hydrogen, phosphate and phosphate salts. Sodium and potassium are example counterions for the phosphate salts. R1, R2, R3, R4, R5 and R6 moieties are independently selected from hydrocarbyl group having 3 to 23 carbons, C ³ -C represented by ^23. For clarity, it will be explained that when a remainder is selected "independently of" a specific group that has several members, it should be understood that the member chosen for the first remainder has no impact or limits in any way the election of the selected member for the second rest. The carbon atoms to which R1, R3, R5 and R6 are attached are asymmetric and, therefore, may exist in the stereochemistry R or S. In one embodiment all those carbon atoms have the R stereochemistry, while in another embodiment embodiment all those carbon atoms have the stereochemistry S.

"Hydrocarbyl" refers to a chemical moiety formed entirely of hydrogen and carbon, where the arrangement of the carbon atoms may be straight or branched chain, non-cyclic or cyclic, and the bond between the adjacent carbon atoms may be by bonds completely, that is, to provide a saturated hydrocarbyl, or there may be double or triple bonds present between any two adjacent carbon atoms, that is, to provide an unsaturated hydrocarbyl, and the number of carbon atoms in the hydrocarbyl group is between 3 and 24 carbon atoms. The hydrocarbyl may be an alkyl, where the representative straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl and the like, including undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, etc .; while branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. The saturated cyclic hydrocarbyls Representative include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like; while unsaturated cyclic hydrocarbyls include cyclopentenyl and cyclohexenyl, and the like. The unsaturated hydrocarbyls contain at least one double or triple bond between adjacent carbon atoms (referred to as an "alkenyl" or "alkynyl", respectively, if the hydrocarbyl is non-cyclic, and cycloalkenyl and cycloalkynyl, respectively, if the hydrocarbyl is at least partially cyclic). Representative straight chain and branched alkenyls include ethylene, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, , 3-dimethyl-2-butenyl, and the like; while representative straight-chain and branched alkynyl include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1 -pentynyl, 2-pentynyl, 3-methyl-butynyl, and the like.

The adjuvant of formula (I) can be obtained by synthetic methods known in the art, for example, the synthetic methodology described in the PCT international publication number WO 2009/035528, as well as the publications identified in WO 2009/035528. Some of the adjuvants can also be obtained commercially. A preferred adjuvant is product No. 699800 as identified in the Avanti Polar Lipids catalog, Alabaster, to L, see E1 in combination with E10, below.

In different embodiments of the description, the adjuvant has the chemical structure of formula (I), but residues A1, A2, R1, R2, R3, R4, R5 and R6 are selected from subsets of the options previously provided for these residues, where these subsets are identified next by E1, E2, etc.

In certain options, each of E2 to E10 is combined with the E1 embodiment, and / or the hydrocarbyl groups of E2 to E9 are alkyl groups, preferably straight chain alkyl groups.

The adjuvant of formula (I) can be formulated in a pharmaceutical composition, optionally with an adjuvant, each as described below. In this regard, reference is made to U.S. Patent Publication. No. 2008/0131466 which provides formulations, e.g. eg, aqueous formulation (AF) and stable emulsion formulations (SE) for the GLA adjuvant, where these formulations can be used for any of the adjuvants of formula (I).

An adjuvant can be administered with the virus of the invention as a single composition, or it can be administered before, simultaneously with or after administration of the recombinant virus of the invention. The immunogen and the adjuvant can be packaged and delivered in the same vial or can be packaged in separate vials and mixed before use. The immunogen and the adjuvant are typically packaged with a label indicating the intended therapeutic application. If the immunogen and the adjuvant are packaged separately, the package typically includes instructions for mixing before use. The choice of an adjuvant and / or vehicle depends on the stability of the vaccine containing the adjuvant, the route of administration, the dosage schedule, the efficacy of the adjuvant for the species being vaccinated and, in humans, a pharmaceutically acceptable adjuvant. it is one that has been approved or can be approved for human administration by the relevant regulatory bodies. For example, Freund's complete adjuvant is not suitable for human administration. Alum, MPL and QS21 are preferred. Optionally, two or more different adjuvants can be used simultaneously, such as alum with MPL, alum with QS21, MPL with QS21 and alum, QS21 and MPL together. In addition, incomplete Freund's adjuvant (Chang et al., Advanced Drug Delivery Reviews 32, 173-186 (1998)), optionally in combination with either alum, QS21 and MPL and all combinations thereof can be used.

Pharmaceutical compositions and kits

Also contemplated herein are pharmaceutical compositions and kits containing a virus provided herein and one or more components. The pharmaceutical compositions can include viral vector particles as provided herein and a pharmaceutical carrier. The kits may include the pharmaceutical compositions and / or combinations provided herein, and one or more components, such as instructions for use, a device for administering a compound to a subject and a device for delivering a compound to a subject.

Herein are provided pharmaceutical compositions containing viral particles as provided herein and a suitable pharmaceutical carrier. The pharmaceutical compositions provided herein may be in different forms, e.g. eg, in solid, liquid, powder, aqueous or lyophilized form. Examples of suitable pharmaceutical carriers are known in the art. Said vehicles and / or additives can be formulated by conventional methods and can be administered to the subject in a suitable dose. Stabilizing agents, such as lipids, nuclease inhibitors, polymers and chelating agents can preserve degradation compositions within the body.

The viral vector particles provided herein can be packaged as kits. The kits may optionally include one or more components, such as instructions for use, devices and additional reagents, and components, such as tubes, containers and syringes for practicing the methods. Example kits may include the viruses provided herein, and may optionally include instructions for use, a device for detecting a virus in a subject, a device for administering the virus to a subject and a device for delivering a compound to a subject. subject.

Kits comprising polynucleotides that encode a gene of interest (typically an antigen) are also contemplated herein. The kit can include at least one plasmid encoding packaging components of the virus and the vector encoding the E2 glycoprotein variant of the Sindbis virus. Some kits will contain at least one plasmid encoding virus packaging components, a vector encoding the E2 glycoprotein variant of the Sindbis virus and a vector encoding at least one DC maturation factor. Kits comprising a viral vector encoding a sequence of interest (typically an antigen) and optionally, a polynucleotide sequence encoding a DC maturation factor are also contemplated herein. In some kits, the kit includes at least one plasmid encoding virus packaging components and a vector encoding the E2 glycoprotein variant of the Sindbis virus.

A kit can also contain instructions. The instructions typically include a tangible expression describing the virus and, optionally, other components included in the kit, and methods of administration, including methods for determining the proper condition of the subject, the appropriate amount of dose and the appropriate method of administration. , to administer the virus. The instructions may also include a guide for tracking the subject during the treatment time.

The kits provided herein may also include a device for delivering a virus to a subject. Any of a variety of devices known in the art can be included to administer drugs or vaccines in kits provided herein. Exemplary devices include, but are not limited to a hypodermic needle, an intravenous needle, a catheter, a needleless injection device, an inhaler and a liquid dispenser, such as an eye dropper. Typically, the device for administering a kit virus will be compatible with the kit virus; for example, a needleless injection device, such as a high-pressure injection device, may be included in kits with viruses not damaged by high-pressure injection, but is typically not included in kits with viruses damaged by injection of high-pressure injection. high pressure. The kits provided herein may also include a device for delivering a compound, such as a DC activator or stimulator, to a subject. Any of a variety of devices known in the art can be included to administer medicaments to a subject in the kits provided herein. Exemplary devices include a hypodermic needle, an intravenous needle, a catheter, a needleless injection, but are not limited to a hypodermic needle, an intravenous needle, a catheter, a needleless injection device, an inhaler and a needle dispenser. liquid such as an eye dropper. Typically, the device for administering the compound of the kit will be compatible with the method of desired administration of the compound.

Example realizations

Methods for generating particles of pseudotyped lentiviral vectors

In some embodiments of the disclosure, a method for generating a pseudotyped lentiviral vector particle comprises:

(a) cultivating in a culture medium comprising kifunensine a virus packaging cell comprising:

(1) a lentiviral vector genome comprising a polynucleotide encoding an exogenous antigen, (2) a polynucleotide encoding an alphavirus glycoprotein that preferentially binds to dendritic cells expressing DC-SIGN, and

(3) a polynucleotide that encodes a Vpx protein or a Vpr protein that retains the inhibitory activity of SAMHD1; Y

(b) isolating a pseudotyped lentiviral vector particle that preferentially binds to dendritic cells expressing DC-SIGN.

In specific aspects, the alphavirus glycoprotein is an E2 glycoprotein of the Sindbis virus.

In specific aspects, the inhibitor of SAMHD1 is a Vpx protein, p. eg, a SIVmac Vpx protein, a SIVsm protein, an SIVrcm or an HIV-2 Vpx protein. In specific aspects, the SAMHD1 inhibitor is an antibody or fragment thereof. In specific aspects, the SAMHD1 inhibitor is a Vpr protein with inhibitory capacity of SAMHD1, for example, a SIVdeb Vpr protein or a SIVmus Vpr protein.

In some embodiments of the disclosure, a method for generating a pseudotyped lentiviral vector particle comprises:

(a) cultivating in a culture medium comprising kifunensine a virus packaging cell comprising:

(1) a lentiviral vector genome comprising a polynucleotide encoding an exogenous antigen,

(2) a polynucleotide encoding a Sindbis E2 glycoprotein that preferentially binds to dendritic cells expressing DC-SIGN, and

(3) a polynucleotide that encodes a Vpx protein or a Vpr protein that retains the inhibitory activity of SAMHD1; Y

(b) isolating a pseudotyped lentiviral vector particle that preferentially binds to dendritic cells expressing DC-SIGN.

In specific aspects, glycoprotein E2 is 90% identical to SEQ ID NO: 30 [SIN-Var1]. In some aspects, (i) residue 160 of glycoprotein E2 is absent or is an amino acid other than glutamic acid, (ii) one or more of residues 70, 76 or 159 of the glycoprotein E2 variant is a non-basic residue , and (iii) the E2 glycoprotein variant is not part of a fusion protein with the E3 glycoprotein of the Sindbis virus. In some aspects, glycoprotein E2 is SEQ ID NO: 30 [SIN-Var1].

In specific aspects, the Vpx protein comprises an amino acid sequence that is at least 80% identical to SIVmac Vpx (SEQ ID NO: 44).

In specific aspects, the Vpx protein comprises at least an 80% amino acid sequence identical to SIVmac Vpx (SEQ ID NO: 44), SIVsm Vpx (SEQ ID NO: 45), SIVrcm Vpx (SEQ ID NO: 46), or HIV -2 Vpx (SEQ ID NO: 47). In specific aspects, the Vpx protein comprises at least 90% amino acid sequence identical to SIVmac Vpx (SEQ ID NO: 44), SIVsm Vpx (SEQ ID NO: 45), SIVrcm Vpx (SEQ ID NO: 46) or HIV - 2 Vpx (SEQ ID NO: 47).

In specific aspects, the Vpr protein of the pseudotyped lentiviral vector particle or lentiviral vector packaging system of any of the preceding embodiments, comprises an amino acid sequence at least 80% identical to SIVdeb Vpr (SEQ ID NO: 48) or SIVmus Vpr (SEQ ID NO: 49). In specific aspects, the Vpr protein of the pseudotyped lentiviral vector particle or lentiviral vector packaging system of any of the above embodiments comprises at least 90% amino acid sequence identical to SIVdeb Vpr (SEQ ID NO: 48) or SIVmus Vpr (SEQ ID NO: 49).

In specific aspects, the antigen is a tumor-specific antigen or a virus-specific antigen. In some aspects, the tumor specific antigen is selected from the group consisting of NY-ESO-1, MAGE, p. eg, MAGE-A3 and MAGE-A1, MART-1 / Melan-A, BAGE, RAGE, gp100, gp75, mda-7, tyrosinase, tyrosinase-related protein, p. eg, TRP2, renal cell carcinoma antigen, 5T4, SM22-alpha, carbonic anhydrase I, carbonic anhydrase IX (also known as G250), HIF-1alpha, HIF-2alpha, VEGF, prostate-specific membrane antigen ( PSMA), prostate specific antigen (PSA), prostatic acid phosphates, prostate epithelial antigen with six transmembrane domains (STEAP), NKX3.1, telomerase enzyme, survivin, mesothelin, mutated ras, bcr / abl rearrangement, Her2 / neu, mutated p53, wild type p53, cytochrome P4501B1, N-acetylglucosaminyltransferase-V, human papillomavirus E6 protein, human papillomavirus E7 protein, carcinoembryonic antigen, Merkel cell virus T antigen oncoproteins and alpha-fetoprotein. In some aspects, the virus-specific antigen is an HIV antigen, an SIV antigen, an adenovirus antigen, an enterovirus antigen, a coronavirus antigen, a calicivirus antigen, a distemper virus antigen, an antigen from Ebola virus, a flavivirus antigen, a hepatitis virus antigen, a herpesvirus antigen, an infectious peritonitis virus antigen, an influenzavirus antigen, a leukemia virus antigen, a Marburg virus antigen , an orthomyxovirus antigen, a papillomavirus antigen, a parainfluenza virus antigen, a paramyxovirus antigen, a parvovirus antigen, a pestivirus antigen, a picornavirus antigen, a poliovirus antigen, a variola virus antigen, a rabies virus antigen, a reovirus antigen, a retrovirus antigen or a rotavirus antigen.

In specific aspects, the genome of the lentiviral vector further comprises a nucleotide sequence encoding a second antigen. In specific aspects, the first and second antigen are expressed as a fusion protein comprising a peptide A2 of self-cleavage between the two antigens. In some aspects, the self-cleaving peptide A2 comprises the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 57. In some aspects, the first antigen is NY-ESO-1 and the second antigen is MAGE-A3.

In specific aspects, kifunensine is present in the culture medium in a concentration of about 0.1 pg / ml to about 10 mg / ml. In some aspects, kifunensine is present in the culture medium at a concentration of about 0.25 pg / ml to about 2 pg / ml. In some aspects, kifunensine is present in the culture medium in a concentration of about 0.01 pg / ml to about 1 pg / ml.

In specific aspects, the virus packaging cell also comprises:

(i) a polynucleotide comprising the gag and pol genes; Y

(ii) a polynucleotide encoding a rev. In some aspects, the polynucleotide encoding the Vpx protein is in the same or different plasmid as the polynucleotide encoding the rev protein, or the polynucleotide comprising the gag and pol genes. In some aspects, the gag and pol genes are optimized human codons and comprise a non-optimized window around position 1228 to 1509 of SEQ ID NO: 54. In some aspects, the polynucleotide comprising the gag and pol genes lacks a response element to rev (RRE). In some aspects, the pol gene encodes an inactive integrase enzyme. In some aspects, the integrase enzyme has a D64V mutation.

In specific aspects, the genome of the lentiviral vector is obtained from HIV-1.

In specific aspects, the genome of the lentiviral vector of the pseudotyped lentiviral vector particle or packaging system of any of the above embodiments is capable of integration.

In specific aspects, the genome of the lentiviral vector of the pseudotyped lentiviral vector particle or the packaging system of any of the foregoing embodiments is of defective integration or poor integration. For example, the lentiviral vector can be integrated with a frequency at least 10 times (eg, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times, at least 300 times, at least 350 times, at least 400 times, at least 450 times, at least 500 times, at least 550 times, at least 600 times, at least 650 times, at least 700 times, at least 750 times, at least 800 times, at least 850 times, at least 900 times, at least 950 times, or at least 1000 times) less effective in integration than a competent integration viral vector. In exemplary embodiments, the lentiviral vector can be at least about 20 times to about 100 times less effective at integration.

In specific aspects, the genome of the lentiviral vector has an inactivated 3 'long terminal repeat (LTR) or a 3' long terminal repeat (LTR) autoinctivating. In some aspects, the genome of the lentiviral vector comprises an U3 element that lacks at least one enhancer sequence, a TATA box, an Sp1 site, an NK-kappa B site or a polypurine tract (PPT). In some aspects, the pol gene encodes an inactive integrase enzyme and the genome of the lentiviral vector lacks a functional polypurine tract (PPT).

In specific aspects, the genome of the lentiviral vector comprises the nucleotide sequence of any one of SEQ ID NO: 21 [SIN vector], 22 [vector 703] or 23 [vector 704].

In specific aspects, the genome of the lentiviral vector further comprises a nucleotide sequence that encodes a dendritic cell stimulation / maturation factor. In some aspects, the dendritic cell stimulation / maturation factor is selected from the group consisting of GM-CSF, IL-2, IL-4, IL-6, IL-7, IL-15, IL-21, IL- 23, TNFa, B7.1, B7.2, 4-1BB, CD40 ligand and drug-induced CD40.

In specific aspects, the nucleotide sequence encoding an antigen is operatively linked to a promoter selected from the group consisting of the human ubiquitin-C (UbiC) promoter, the cytomegalovirus immediate early promoter (CMV), the Rous sarcoma (RSV), and the promoter sensitive to tetracyclines. In some aspects, the promoter is an introns deficient promoter. In some aspects, the introns deficient promoter is a UbiC.

In specific aspects, a lentiviral vector particle produced by the method of paragraph [0206], [0210] or [0218] is provided.

In specific aspects, a lentiviral vector particle produced by the method of paragraph [0216] is provided.

Compositions comprising particles of pseudotyped lentiviral vectors.

In some or any of the embodiments of the disclosure, there is provided a composition comprising pseudotyped lentiviral vector particles comprising (a) a Vpx protein or a Vpr protein that retains the inhibitory activity of SAMHD1, (b) an exogenous polynucleotide encoding an antigen, and (c) a plurality of envelope glycoproteins that preferentially bind to cells expressing DC-SIGN, wherein the composition is more highly mannosylated compared to a control composition of the same pseudotyped lentiviral vector particles prepared in absence of a mannosidase inhibitor. For example, said highly-mannosylated composition is characterized by containing envelope glycoproteins that are more sensitive to EndoH than the envelope glycoproteins of a control composition prepared in the absence of a mannosidase inhibitor. As another example, said highly mannosylated composition is characterized by containing envelope glycoproteins having a greater amount of EndoH sensitive glycan in comparison to the envelope glycoproteins of a control composition prepared in the absence of a mannosidase inhibitor. It is understood that the control composition will contain about the same number of particles of pseudotyped lentiviral vectors comprising envelope glycoproteins having the same amino acid sequence (s). It is also understood that although the description is mainly focused on the glycoprotein E2 of the alphavirus, which is responsible for the recognition and binding to dendritic cells, and their highly-mannosylated versions, the alphavirus envelope also contains E1 glycoproteins that are susceptible to mannosylation. high. Thus, references to envelope glycoproteins and highly-mannosylated envelope glycoproteins include references to E2 and / or E1 glycoproteins of highly-mannosylated alphaviruses, specifically the highly-syn- missiated E2 and / or E1 glycoproteins of Sindbis. The high mannosylation of other types of virus envelopes, specifically enveloped by retroviruses, also improves the recognition of dendritic cells.

In some embodiments of the disclosure, there is provided a composition comprising pseudotyped lentiviral vector particles comprising (a) a Vpx protein or a Vpr protein that retains the inhibitory activity of SAMHD1, (b) an exogenous polynucleotide encoding an antigen, and (c) a plurality of Sindbis E2 glycoproteins that preferentially bind to cells expressing DC-SIGN, wherein the composition is more highly mannosylated in comparison to a control composition of pseudotyped lentiviral vector particles prepared in the absence of an inhibitor of mannosidase A substantial number of E2 glycoproteins in said compositions are more highly mannosylated, e.g. eg, more sensitive to EndoH, than E2 glycoproteins in control compositions prepared in the absence of a mannosidase inhibitor.

EndoH is a specialized endoglycosidase that will only cleave N-linked glycosylation with high mannose content (see Figure 3A), p. g., glycans Man5-9; Man3-4 glycans are resistant to EndoH. In contrast, PNGase F will cleave all types of glycosylation, including the low mannose glycans <Man5. When viral particles are produced in the presence of a mannosidase I inhibitor, such as kifunensine, the glycoproteins in the viral envelope will contain more glycans with high mannose content that are susceptible to being cleaved by EndoH (ie, they will be more "sensitive"). EndoH "). One way to detect the presence of glycans with high mannose content in a composition of viral particles is to determine the molecular weight of the envelope glycoproteins by electrophoresis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after treatment with EndoH . This "gel shift" test is illustrated in Example 3. Samples of the viral particles and control compositions of the same viral particles prepared in the absence of mannosidase inhibitor can be carried on an SDS-PAGE gel at 10 ° C. % and immunoblot with antibody against Sindbis glycoproteins The envelope of the virus produced in the presence of mannosidase I inhibitors is more sensitive to EndoH and, therefore, moves faster in the gel (a greater distance) after treatment with EndoH , compared to the control virus Therefore, the presence of high mannose glycoprotein can be measured using a gel change test after treatment with EndoH.

The control virus may be partially sensitive to EndoH treatment due to the presence of glycosylation sites in the Sindbis glycoproteins that are normally high in mannose because they are internal and are not exposed to mannosidase I during production. Therefore, when carried to 10% SDS-PAGE, the control virus may show some change in molecular weight after treatment with EndoH, due to the EndoH cleavage of said internal glycans with high mannose content. However, this change may only be about 35% of the change observed when the control virus is treated with PNGase F, which cleaves all glycans.

Therefore, in specific aspects, the amount of EndoH sensitive glycan is detected by determining the molecular weight of the Sindbis glycoproteins by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after treatment with EndoH. A greater amount of endoH sensitive glycan is detected in the composition, which indicates that the composition of the viral particles is highly mannosylated, when the EndoH treatment results in the band corresponding to the envelope glycoproteins (E2 and / or E1) on a 10% SDS-PAGE gel changes to a lower molecular weight as compared to the control envelope glycoproteins, determined by visual inspection.

In some illustrative embodiments, the molecular weight of said envelope glycoproteins after treatment with EndoH has completely changed, and is substantially the same as the molecular weight of said envelope glycoproteins after treatment with PNGaseF. In other illustrative embodiments, the molecular weight of said envelope glycoproteins after treatment with EndoH has changed approximately 90% or more of the distance between (a) envelope glycoproteins not treated with endoglycosidase, and (b) envelope glycoproteins treated with PNGase F. In other illustrative embodiments, the molecular weight of said envelope glycoproteins after treatment with EndoH has changed approximately 90% or more of the distance between (a) envelope glycoproteins not treated with endoglycosidase, and (b) glycoproteins of the envelope treated with PNGase F. In still other illustrative embodiments, the molecular weight of said envelope glycoproteins has changed about 40% or more, or preferably about 45% or more, or about 50% or more, or about 55% or more, about 60% or more, or about 65% or more, or about 70% or more, or about 75% or more, or about approximately 80% or more, or approximately 85% or more of the distance between (a) envelope glycoproteins not treated with endoglycosidase, and (b) envelope glycoproteins treated with PNGase F. As indicated above, reference to glycoproteins of the envelope specifically includes reference to glycoproteins E2 and / or E1 of Sndbis.

In specific aspects, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at less 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the envelope glycoproteins in said composition, e.g. For example, the E2 and / or E1 glycoproteins of Sindbis in said composition have a greater amount of EndoH sensitive glycan compared to the control envelope glycoproteins having the same sequence (s) of amino acids in a control composition of pseudotyped lentiviral vector particles prepared in the absence of a mannosidase inhibitor.

In some embodiments of the disclosure, a composition comprises particles of pseudotyped lentiviral vectors comprising (a) a SAMHD1 inhibitor, (b) a lentiviral genome comprising a sequence of interest, and (c) a envelope glycoprotein that binds preferentially to cells expressing DC-SIGN, wherein at least 80% of the N-linked glycans in said composition comprise a Mang structure. In specific aspects, the inhibitor of SAMHD1 is a Vpx protein, p. eg, a SlVmac Vpx protein, a SIVsm Vpx protein, a SlVrcm Vpx protein or an HIV-2 Vpx protein. In specific aspects, the SAMHD1 inhibitor is an antibody or fragment thereof. In specific aspects, the inhibitor of SAMHD1 is a Vpr protein with inhibitory capacity of SAMHD1, p. eg, a SlVdeb Vpr protein or a SlVmus Vpr protein. In specific aspects, the sequence of interest encodes a protein or a nucleic acid molecule, such as a siRNA, microRNA, a self-complementary double-stranded RNA in which the complementary region is greater than about 20 ribonucleotides in length, or an RNA that is complementary of a messenger RNA, where the binding of said complementary RNA (antiparallel) to messenger RNA blocks its ability to be translated into protein. In some cases, the sequence of interest encodes an antigen against which an immune response is desired. In specific aspects, the sequence of interest encodes a tumor antigen or antigens of an infectious disease (eg, of agents such as HIV, HSV, HCV, HPV, malaria or tuberculosis). In specific aspects, multiple sequences of interest are included in a single vector.

In some embodiments of the disclosure, a composition comprises particles of pseudotyped lentiviral vectors comprising (a) a Vpx protein, (b) an exogenous polynucleotide encoding an antigen, and (c) a envelope glycoprotein that preferentially binds to dendritic cells expressing DC-SIGN, wherein at least 80% of the glycans linked to N in said composition comprise a Mang structure.

In specific aspects, the Vpx protein comprises an amino acid sequence that is at least 80% identical to the SIVmac Vpx protein (SEQ ID NO: 44).

In specific aspects, the Vpx protein comprises at least an 80% amino acid sequence identical to SIVmac Vpx (SEQ ID NO: 44), SIVsm Vpx (SEQ ID NO: 45), SIVrcm Vpx (SEQ ID NO: 46) or HIV- 2 Vpx (SEQ ID NO: 47). In specific aspects, the Vpx protein comprises at least 90% amino acid sequence identical to SIVmac Vpx (SEQ ID NO: 44), SIVsm Vpx (SEQ ID NO: 45), SIVrcm Vpx (SEQ ID NO: 46) or HIV- 2 Vpx (SEQ ID NO: 47).

In specific aspects, the Vpr protein of the pseudotyped lentiviral vector particle or lentiviral vector packaging system or any of the above embodiments comprises at least an 80% amino acid sequence identical to SIVdeb Vpr (SEQ ID NO: 48) or SIVmus Vpr (S ^e Q ID NO: 49). In specific aspects, the Vpr protein of the pseudotyped lentiviral vector particle or lentiviral vector packaging system of any of the above embodiments comprises at least 90% amino acid sequence identical to SIVdeb Vpr (SEQ ID NO: 48) or SIVmus Vpr (SEQ ID NO: 49).

In specific aspects, the pseudotyped lentiviral vector particle infects dendritic cells expressing DC-SIGN with an in vitro transduction efficiency of at least 1%, or at least 5%, or at least 10%, or at least 20%. In specific aspects, the glycoprotein is an E2 glycoprotein of the Sindbis virus. In some aspects, glycoprotein E2 has at least 90% identity with SEQ ID NO: 30 [SIN-Var1]. In some aspects, (i) residue 160 of glycoprotein E2 is absent or is an amino acid other than glutamic acid, (ii) one or more of residues 70, 76 or 159 of the glycoprotein E2 variant is a non-glycoprotein basic, and (iii) the E2 glycoprotein variant is not part of a fusion protein with the E3 glycoprotein of the Sindbis virus.

In specific aspects, the antigen is a tumor-specific antigen or a virus-specific antigen. In some aspects, the tumor specific antigen is selected from the group consisting of NY-ESO-1, MAGE, MAGE-A3 and MAGE-A1, MART-1 / Melan-A, BAGE, RAGE, gp100, gp75, mda- 7, tyrosinase, protein related to tyrosinase, p. eg, TRP2, renal cell carcinoma antigen, 5T4, SM22-alpha, carbonic anhydrase I, carbonic anhydrase IX (also known as G250), HIF-1alpha, HIF-2alpha, VEGF, prostate-specific membrane antigen ( PSMA), prostate specific antigen (PSA), prostatic acid phosphates, prostate epithelial antigen with six transmembrane domains (STEAP), NKX3.1, telomerase enzyme, survivin, mesothelin, mutated ras, bcr / abl rearrangement, Her2 / neu, mutated p53, wild type p53, cytochrome P4501B1, N-acetylglucosaminyltransferase-V, human papillomavirus E6 protein, human papillomavirus E7 protein, carcinoembryonic antigen, Merkel cell virus T antigen oncoproteins and alpha-fetoprotein. In some aspects, the virus-specific antigen is an HIV antigen, an SIV antigen, an adenovirus antigen, an enterovirus antigen, a coronavirus antigen, a calicivirus antigen, a distemper virus antigen, an antigen from Ebola virus, a flavivirus antigen, a hepatitis virus antigen, a herpesvirus antigen, an infectious peritonitis virus antigen, an influenzavirus antigen, a leukemia virus antigen, a Marburg virus antigen , an orthomyxovirus antigen, a papillomavirus antigen, a parainfluenza virus antigen, a paramyxovirus antigen, a parvovirus antigen, a pestivirus antigen, a picornavirus antigen, a poliovirus antigen, a smallpox virus antigen, a rabies virus antigen, a reovirus antigen, a retrovirus antigen or a rotavirus antigen.

In specific aspects, the genome of the lentiviral vector further comprises a nucleotide sequence encoding a second antigen. In specific aspects, the first and second antigen are expressed as a fusion protein comprising a peptide A2 of self-cleavage between the two antigens. In some aspects, the self-cleaving peptide A2 comprises the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 57. In some aspects, the first antigen is MAGE-A3 and the second antigen is NY-ESO-1.

In specific aspects, the genome of the lentiviral vector is obtained from HIV-1.

In specific aspects, the genome of the lentiviral vector of the pseudotyped lentiviral vector particle or packaging system of any of the above embodiments is capable of integration.

In specific aspects, the genome of the lentiviral vector of the pseudotyped lentiviral vector particle or packaging system of any of the previous embodiments is of poor integration or defective integration.

In specific aspects, the genome of the lentiviral vector has an inactivated 3 'long terminal repeat (LTR) or a self-activating 3' long terminal repeat (LTR). In some aspects, the genome of the lentiviral vector comprises an U3 element that lacks at least one enhancer sequence, a TATA box, an Sp1 site, an NK-kappa B site or a polyprin (PPT) stretch.

In specific aspects, the genome of the lentiviral vector comprises the nucleotide sequence of any of SEQ ID NO: 21 [SIN vector], 22 [vector 703] or 23 [vector 704].

In specific aspects, the genome of the lentiviral vector further comprises a nucleotide sequence that encodes a maturation / stimulation factor of dendritic cells. In some aspects, the maturation / stimulation factor of dendritic cells is selected from the group consisting of GM-CSF, IL-2, IL-4, IL-6, IL-7, IL-15, IL-21, IL -23, TNFa, B7.1, B7.2, 4-1BB, CD40 ligand and CD40 inducible by drug.

In specific aspects, the nucleotide sequence encoding an antigen is operatively linked to a promoter selected from the group consisting of the human ubiquitin-C (UbiC) promoter, the cytomegalovirus immediate early promoter (CMV), the Rous sarcoma (RSV) and the promoter sensitive to tetracyclines. In some aspects, the promoter has been modified to be deficient in introns.

In some aspects, the pseudotyped lentiviral vector particles comprise a Rev protein.

In specific aspects, the particles of pseudotyped lentiviral vectors have a UI of at least 105 / ml. In specific aspects, the composition further comprises an immunostimulatory agent.

In specific aspects, the composition further comprises an adjuvant. For example, as indicated above, adjuvants include alum or monophosphoryl lipid A (MPL) 3 De-O-acylated. The classes of adjuvants described herein include (a) aluminum salts, (b) oil-in-water emulsion formulations, optionally with or without other specific immunostimulatory agents, such as muramyl peptides or other components of the bacterial cell wall. , (c) saponin adjuvants, including ISCOM (immunostimulatory complexes) and ISCOMATRIX; (d) Freund's complete adjuvant (CFA) and incomplete Freund's adjuvant (IFA); (e) cytokines; and (f) adjuvants of formula (I). Within formula (I), a preferred adjuvant is product No. 699800 as identified in the Avanti Polar Lipids catalog, Alabaster, AL, see E1 in combination with E10, where (i) A1 is phosphate or phosphate salt and A2 is hydrogen and (ii) R1, R3, R5 and R6 are undecyl and R2 and R4 are tridecyl. In specific aspects, envelope glycoproteins also bind to cells expressing mouse SIGNR1.

In specific aspects, the pseudotyped lentiviral vector particles also more efficiently transduce cells expressing mouse SIGNR1 compared to cells that do not express mouse SIGNR1.

In some embodiments of the disclosure, the pseudotyped lentiviral vector particles mentioned above are for use in a method of treating or preventing a disease or disorder in a patient. In specific aspects, the disease or disorder is a cancer, an autoimmune disease or an infection, for example, a viral infection, a bacterial infection, a fungal infection or a parasitic infection.

Viral vector particles comprising a Vpx protein.

In some embodiments of the disclosure, a pseudotyped lentiviral vector particle capable of targeting an expressing cell comprising:

(a) an unnatural envelope glycoprotein;

(b) a lentiviral vector genome comprising an exogenous polynucleotide of interest; Y

(c) a Vpx protein or another inhibitor of SAMHD1.

In some aspects, the pseudotyped lentiviral vector particle further comprises a Rev protein.

In some embodiments of the disclosure, a lentiviral vector packaging system is provided to produce a pseudotyped lentiviral vector particle, comprising:

(i) a first polynucleotide that encodes an unnatural envelope glycoprotein;

(ii) a second polynucleotide comprising gag and pol genes;

(iii) a third polynucleotide encoding a rev protein;

(iv) a fourth polynucleotide that encodes a Vpx protein or another inhibitor of SAMHD1; Y

(v) a lentiviral vector genome comprising an exogenous polynucleotide of interest, wherein two or more polynucleotides are on the same plasmid or on different plasmids. In specific aspects, the polynucleotide of (iv) is in the same plasmid as any one or more of the polynucleotides of (i), (ii), (iii) or (v).

In specific aspects, the packaging cell is selected from the group consisting of 293, 293T, HeLa, D17, MDCK, BHK and Cf2Th cells.

In specific aspects, the Vpx protein of the pseudotyped lentiviral vector particle or the lentiviral vector packaging system of any of the above embodiments comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 44 (SIVmac) , optionally a SIVmac Vpx protein (SEQ ID NO: 44), a SIVsm Vpx protein (SEQ ID NO: 45), a SIVrcm Vpx protein (SEQ ID NO: 46) or an HIV-2 VPX protein (SEQ ID NO: 47) ). In specific aspects, the Vpx protein of the pseudotyped lentiviral vector particle or lentiviral vector packaging system of any of the above embodiments comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 44 (SIVmac), optionally a SIVmac Vpx protein (SEQ ID NO: 44), SIVsm Vpx protein (SEQ ID NO: 45), SIVrcm Vpx protein (SEQ ID NO: 46), or an HIV-2 VPX protein (SEQ ID NO: 47).

In specific aspects, the Vpr protein of the pseudotypic lentiviral vector particle or lentiviral vector packaging system of any of the above embodiments comprises at least an 80% amino acid sequence identical to SIVdeb Vpr (SEQ ID NO: 48) or SIVmus Vpr (SEQ ID NO: 49). In specific aspects, the Vpr protein of the pseudotyped lentiviral vector particle or lentiviral vector packaging system of any of the above embodiments comprises at least 90% amino acid sequence identical to SIVdeb Vpr (SEQ ID NO: 48) or SIVmus Vpr (SEQ ID NO: 49).

In specific aspects, the genome of the lentiviral vector of the pseudotyped lentiviral vector particle or lentiviral vector packaging system of any of the above embodiments is obtained from HIV-1 or MLV. In specific aspects, the genome of the lentiviral vector of the particle of the pseudotyped lentiviral vector or packaging system of any of the above embodiments is capable of integration.

In specific aspects, the genome of the lentiviral vector of the pseudotyped lentiviral vector particle or packaging system of any of the above embodiments is not integral.

In specific aspects, the unnatural envelope glycoprotein of the pseudotyped lentiviral vector particle or packaging system of any of the above embodiments is selected from the group consisting of an alphavirus glycoprotein, which includes Sindbis glycoprotein E2, VEE glycoprotein E2. , rhabdovirus glycoprotein or vesiculovirus, which includes the glycoprotein VSV-G, arenavirus glycoprotein, coronavirus glycoprotein, paramyxovirus glycoprotein, flavirvirus glycoprotein, orthomyxovirus glycoprotein and baculovirus glycoprotein, preferably an alphavirus such as Sindbis virus.

In specific aspects, the unnatural envelope glycoprotein of the pseudotyped lentiviral vector particle or packaging system of any of the foregoing embodiments is an E2 glycoprotein of the Sindbis virus comprising at least an 80% amino acid sequence identical to SEQ ID NO. : 30 [SINVar1].

In specific aspects, the unnatural envelope glycoprotein of the pseudotyped lentiviral vector particle or packaging system of any of the foregoing embodiments is preferably linked to a cell expressing SAMHD1. In some aspects, the cell expressing SAMHD1 is a myeloid cell, optionally a dendritic cell, a monocyte or a macrophage.

In specific aspects, the unnatural envelope glycoprotein of the pseudotyped lentiviral vector particle or packaging system of any of the foregoing embodiments binds preferentially to dendritic cells expressing DC-SIGN.

In specific aspects, the polynucleotide of interest encodes (i) an antigen, (ii) a therapeutic polypeptide, or (iii) an inhibitory oligonucleotide. In some aspects, the polynucleotide of interest encodes an siRNA.

In specific aspects, the polynucleotide of interest encodes a viral, bacterial, fungal, protozoan or cancer antigen.

In specific aspects, the envelope glycoproteins also bind to cells expressing mouse SIGNR1. In specific aspects, the pseudotyped lentiviral vector particles also more efficiently transduce cells expressing mouse SIGNR1 compared to cells that do not express mouse SIGNR1.

In some embodiments of the disclosure, there is provided a method for producing a pseudotyped lentiviral vector particle of any of the preceding embodiments which comprises culturing the packaging system of any of the above embodiments in a culture medium. In some aspects, the culture medium comprises a mannosidase I inhibitor which is kifunensine.

In some embodiments of the disclosure, a composition comprising the pseudotyped lentiviral vector particles of any of the preceding embodiments is provided wherein the vector particles are highly mannosylated.

In some embodiments of the disclosure, a composition comprising the pseudotyped lentiviral vector particles of any of the preceding embodiments is provided wherein at least 80% of the N-linked glycans in said composition comprise a Mang structure.

In some embodiments of the disclosure, there is provided a method for delivering a lentiviral vector genome to a cell expressing SAMHD1, in vitro or in vivo, comprising contacting the cell with the vector particle of any of the above embodiments. In some aspects, the cell expressing SAMHD1 is a dendritic cell, a monocyte or a macrophage.

In some embodiments of the disclosure, there is provided a method for eliciting an immune response or immunizing an individual, comprising administering the vector particle of any of the foregoing embodiments to an individual, preferably a vector particle that preferentially binds to cells. Dendritic cells that express DC-SIGN.

In some embodiments of the disclosure, the pseudotyped lentiviral vector particles mentioned above are for use in a method of treating or preventing a disease or disorder in a patient. In specific aspects, the disease or disorder is a cancer, an autoimmune disease or an infection, for example, a viral infection, a bacterial infection, a fungal infection or a parasitic infection.

Methods of generation of viral vector particles with highly mannosylated envelope glycoproteins In some embodiments of the disclosure, there is provided a method for generating a virus vector particle that preferentially binds dendritic cells expressing DC-SIGN comprising: culturing a virus packaging cell comprising virus component viruses in a culture medium, said components comprising a polynucleotide encoding a envelope glycoprotein that preferentially binds to dendritic cells expressing DC-SIGN, and wherein the culture medium comprises kifunensine in a concentration of about 0.01 pg / ml to about 1 mg / ml, preferably from about 0.1 pg / ml to about 10 pg / ml.

In specific aspects, kifunensine is present in the culture medium in a concentration of about 0.25 pg / ml to about 2 pg / ml.

In specific aspects, kifunensine is present in the culture medium at a concentration of about 0.1 pg / ml to about 10 pg / ml, or from about 0.25 pg / ml to about 2 pg / ml, or about 0.5 pg / ml to around 5 pg / ml.

In specific aspects, the viral particle components comprise a lentiviral vector genome.

In specific aspects, the viral particle infects cells expressing DC-SIGN with a transduction efficiency at least 5 times greater than a viral particle produced in a culture medium lacking kifunensine.

In specific aspects, the virus packaging cell comprises:

(i) a first polynucleotide encoding a envelope glycoprotein;

(ii) a second polynucleotide comprising the gag and pol genes;

(iii) a third polynucleotide encoding a rev protein; Y

(iv) a lentiviral vector genome comprising a fourth polynucleotide encoding an antigen.

In specific aspects, the envelope glycoprotein is an E2 glycoprotein of the Sindbis virus. In some aspects, glycoprotein E2 comprises [SINVar1] or a variant thereof having at least 80% amino acid sequence identity therewith. In some aspects, glycoprotein E2 is 90% identical to SEQ ID NO: 30 [SIN-Var1]. In some aspects, (i) residue 160 of glycoprotein E2 is absent or is an amino acid other than glutamic acid, (ii) one or more of residues 70, 76 or 159 of the glycoprotein E2 variant is a non-glycoprotein basic, and (iii) the E2 glycoprotein variant is not part of a fusion protein with the E3 glycoprotein of the Sindbis virus. In some aspects, glycoprotein E2 is SEQ ID NO: 30 [SIN-Var1].

In specific aspects, the genome of the lentiviral vector is obtained from HIV-1.

In specific aspects, the genome of the lentiviral vector of the particle of the pseudotyped lentiviral vector or packaging system of any of the above embodiments is capable of integration.

In specific aspects, the genome of the lentiviral vector of the particle of the pseudotyped lentiviral vector or packaging system of any of the above embodiments is of poor integration in the defective integration. In some aspects, the pol gene encodes an inactive integrase enzyme and the genome of the lentiviral vector lacks a functional polypurine tract (PPT).

In specific aspects, the virus packaging cell further comprises a polynucleotide that encodes a Vpx protein that retains the inhibitory activity of SAMHD1, optionally comprising at least an 80% amino acid sequence identical to SIVmac Vpx (SEQ ID NO: 44).

In some embodiments of the disclosure, there is provided a composition comprising virus particles having an alpha2 virus glycoprotein E2, wherein at least 80% of the N-linked glycans in said composition comprise a Mang structure.

In specific aspects, alphavirus glycoprotein E2 is a Sindbis E2 glycoprotein that preferentially binds to dendritic cells expressing DC-SIGN.

In some embodiments of the disclosure, there is provided a method for delivering a viral vector genome to a cell expressing DC-SIGN which comprises administering the virus particle or composition of any of the above embodiments.

Additional aspects of the description

A method for generating a pseudotyped lentiviral vector particle comprising:

(a) cultivating in a culture medium comprising kifunensine a virus packaging cell comprising:

(1) a lentiviral vector genome comprising a polynucleotide encoding an exogenous antigen, (2) a polynucleotide encoding a Sindbis E2 glycoprotein that preferentially binds to dendritic cells expressing DC-SIGN, and

(3) a polynucleotide that encodes a Vpx protein or a Vpr protein that retains the inhibitory activity of SAMHD 1; Y

(b) isolating a pseudotyped lentiviral vector particle that preferentially binds to dendritic cells expressing DC-SIGN.

2. The method of embodiment 1, wherein glycoprotein E2 is 90% identical to SEQ ID NO: 30 [SIN-Var1].

3. The method of embodiment 1 or 2, wherein (i) residue 160 of glycoprotein E2 is absent or is an amino acid other than glutamic acid, (ii) one or more of residues 70, 76 or 159 of the E2 glycoprotein variant is a non-basic residue, and (iii) the E2 glycoprotein variant is not part of a fusion protein with E3 glycoprotein Sindbis virus.

4. The method of embodiment 2, wherein glycoprotein E2 is SEQ ID NO: 30 [SIN-Var1].

5. The method of any one of the preceding embodiments, wherein the Vpx protein comprises an amino acid sequence that is at least 80% identical to SIVmac Vpx [SEQ ID NO: 44].

The method of any one of embodiments 1 to 4, wherein the Vpx protein comprises at least 90% amino acid sequence identical to SIVmac Vpx (SEQ ID NO: 44), SIVsm Vpx (SEQ ID NO: 45), SIVrcm Vpx (SEQ ID NO: 46), or HIV-2 Vpx (SEQ ID NO: 47).

The method of any one of embodiments 1 to 4, wherein the Vpx protein comprises at least 90% amino acid sequence identical to SIVdeb Vpr (SEQ ID NO: 48) or SIVmus Vpr (SeQ ID NO: 49).

8. The method of any one of the preceding embodiments, wherein the antigen is a tumor-specific antigen or a virus-specific antigen.

9. The method of embodiment 8, wherein the tumor specific antigen is selected from the group consisting of NY-ESO-1, MAGE, MART-1 / Melan-A, BAGE, RAGE, gp100, gp75, mda-7 , tyrosinase, tyrosinase-related protein, renal cell carcinoma antigen, 5T4, SM22-alpha, carbonic anhydrase I, carbonic anhydrase IX (also known as G250), HIF-1alpha, HIF-2alpha, VEGF, specific membrane antigen of the prostate (PSMA), prostate specific antigen (PSA), prostatic acid phosphates, prostate epithelial antigen with six transmembrane domains (STEAP), NKX3.1, telomerase enzyme, survivin, mesothelin, mutated ras, rearrangement bcr / abl, Her2 / neu, mutated p53, wild type p53, cytochrome P450 1B1, N-acetylglucosaminyltransferase-V, human papilloma virus E6 protein, human papillomavirus E7 protein, carcinoembryonic antigen and alpha-fetoprotein.

10. The method of embodiment 8, wherein the virus-specific antigen is an HIV antigen, an SIV antigen, an adenovirus antigen, an enterovirus antigen, a coronavirus antigen, a calicivirus antigen, an antigen of distemper virus, an Ebola virus antigen, a flavivirus antigen, a hepatitis virus antigen, a herpesvirus antigen, an infectious peritonitis virus antigen, an influenzavirus antigen, a leukemia virus antigen , a Marburg virus antigen, an orthomyxovirus antigen, a papillomavirus antigen, a parainfluenza virus antigen, a paramyxovirus antigen, a parvovirus antigen, a pestivirus antigen, a picornavirus antigen, a poliovirus antigen, a smallpox virus antigen, a rabies virus antigen, a reovirus antigen, a retrovirus antigen or a rotavirus antigen.

11. The method of any one of the preceding embodiments, wherein the genome of the lentiviral vector further comprises a nucleotide sequence encoding a second antigen.

12. The method of embodiment 11, wherein the first and second antigen are expressed as a fusion protein comprising a peptide A2 of self-cleavage between the two antigens.

13. The method of embodiment 12, wherein the self-cleaving peptide A2 comprises the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 57.

The method of any one of embodiments 11 to 13, wherein the first antigen is NY-ESO-1 and the second antigen is MAGE-A3.

15. The method of embodiment 11, wherein the first and second antigens are expressed from a bidirectional promoter.

16. The method of any one of the preceding embodiments, wherein the kifunensine is present in the culture medium in a concentration of about 0.1 pg / ml to about 10 pg / ml.

17. The method of embodiment 16, wherein the kifunensine is present in the culture medium in a concentration of about 0.25 pg / ml to about 2 pg / ml.

18. The method of any one of the preceding embodiments, wherein the virus packaging cell further comprises:

(i) a polynucleotide comprising the gag and pol genes; Y

(ii) a polynucleotide encoding a rev.

19. The method of embodiment 18, wherein the gag and pol genes are human optimized codons and comprise a non-optimized window around position 1228 to 1509 of SEQ ID NO: 54.

20. The method of embodiment 18 or 19, wherein the polynucleotide comprising the gag and pol genes lacks a functional rev response element (RRE).

21. The method of any one of embodiments 18 to 20, wherein the pol gene encodes an inactive integrase enzyme.

22. The method of embodiment 21, wherein the integrase enzyme has a D64V mutation.

The method of any one of embodiments 18 to 22, wherein the polynucleotide encoding the Vpx protein is on the same or different plasmid as the polynucleotide encoding the rev protein, or the polynucleotide comprising the gag and pol genes.

24. The method of any one of the preceding embodiments, wherein the genome of the lentiviral vector is derived from HIV-1.

25. The method of any one of the preceding embodiments, wherein the genome of the lentiviral vector has an inactivated 3 'long terminal repeat (LTR) or a self-activating 3' long terminal repeat (LTR).

26. The method of embodiment 25, wherein the genome of the lentiviral vector comprises an U3 element that lacks at least one enhancer sequence, a TATA box, an Sp1 site, an NK-kappa B site, or a polypurine tract ( PPT).

27. The method of any one of the preceding embodiments, wherein the genome of the lentiviral vector comprises the nucleotide sequence of any of SEQ ID NO: 21, 22 or 23.

28. The method of any one of the preceding embodiments, wherein the genome of the lentiviral vector further comprises a nucleotide sequence that encodes a dendritic cell stimulation / maturation factor.

29. The method of embodiment 28, wherein the dendritic cell stimulation / maturation factor is selected from the group consisting of GM-CSF, IL-2, IL-4, IL-6, IL-7, IL-15. , IL-21, IL-23, TNFa, B7.1, B7.2, 4-1BB, CD40 ligand and drug-induced CD40.

30. The method of any one of the preceding embodiments, wherein the nucleotide sequence encoding an antigen is operatively linked to a promoter selected from the group consisting of the human ubiquitin-C promoter (UbiC), the immediate promoter of cytomegalovirus (CMV), the Rous sarcoma virus promoter (RSV) and the sensitive promoter tetracyclines.

31. The method of embodiment 30, wherein the promoter is an introns deficient promoter.

32. The method of embodiment 31, wherein the introns deficient promoter is a UbiC promoter.

33. The lentiviral vector particle produced by the embodiment of claim 1.

34. The lentiviral vector particle produced by the embodiment of claim 18.

35. A method for generating a pseudotyped lentiviral vector particle comprising:

(a) cultivating in a culture medium comprising kifunensine a virus packaging cell comprising:

(1) a lentiviral vector genome comprising a polynucleotide encoding MAGE-A3 and NY-ESO-1, wherein a polynucleotide encoding a self-cleaving TA2 peptide is placed between the polynucleotide that encodes MAGE-A3 and NY-ESO-1, where the lentiviral genome lacks a polypurine tract (PPT), and where the expression of MAGE-A3 and NY-ESO-1 is controlled by a UbiC promoter that lacks of an intron,

(2) a polynucleotide encoding a Sindbis E2 glycoprotein that preferentially binds to dendritic cells expressing DC-SIGN,

(3) a polynucleotide comprising the human codon optimized gag and pol genes, wherein the polynucleotide lacks a functional rev response element (RRE) and wherein the pol gene encodes an inactive integrase enzyme,

(4) a polynucleotide that encodes a Vpx protein or a Vpr protein that retains the inhibitory activity of SAMHD1; Y

(b) isolating a pseudotyped lentiviral vector particle that preferentially binds to dendritic cells expressing DC-SIGN.

36. A composition comprising pseudotyped lentiviral vector particles comprising (a) a Vpx protein or a Vpr protein that retains the inhibitory activity of SAMHD1, (b) an exogenous polynucleotide encoding an antigen, and (c) a plurality of glycoproteins of envelope that bind preferentially to cells expressing DC-SIGN, wherein said composition is more highly mannosylated compared to a control composition of the same pseudotyped lentiviral vector particles prepared in the absence of a mannosidase inhibitor.

37. The composition of embodiment 36, wherein the envelope glycoproteins are alphavirus glycoproteins.

38. The composition of embodiment 37, wherein the envelope glycoproteins are Sindbis glycoproteins. 39. The composition of any of embodiments 36-38, wherein high mannosylation is characterized as being more sensitive to EndoH than a control composition prepared in the absence of a mannosidase inhibitor. 40. The composition of any of embodiments 36-38, wherein the sensitivity to EndoH is determined by evaluating the molecular weight of the envelope glycoproteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after treatment with EndoH .

41. The composition of any one of embodiments 36-38, wherein the molecular weight of the envelope glycoproteins after treatment with EndoH has changed approximately 45% or more of the distance between (a) the envelope glycoproteins. treated with an endoglycosidase, and (b) the envelope glycoproteins treated with PNGase F.

42. The composition of embodiment 41, wherein the molecular weight of the envelope glycoproteins after treatment with EndoH has changed approximately 70% or more of the distance between (a) the envelope glycoproteins not treated with endoglycosidase, and (b) envelope glycoproteins treated with PNGase F.

43. The composition of embodiment 41, wherein the molecular weight of the envelope glycoproteins after treatment with EndoH has changed approximately 90% or more of the distance between (a) the envelope glycoproteins not treated with endoglycosidase, and (b) envelope glycoproteins treated with PNGase F.

44. The composition of any one of embodiments 36-43, wherein at least 30% of the envelope glycoproteins in said composition have a greater amount of glycan responsive to EndoH compared to the control glycoproteins having the ) same amino acid sequence (s) in a control composition of pseudotyped lentiviral vector particles prepared in the absence of a mannosidase inhibitor.

45. The composition of any one of embodiments 36-44, wherein most of the envelope glycoproteins are highly mannosylated.

46. The composition of any one of embodiments 36-45, wherein the particles of pseudotyped lentiviral vectors are poorly integrated.

47. The composition of any one of embodiments 36-46, wherein the composition comprises a Sindbis E2 glycoprotein.

48. The composition of embodiment 47, wherein glycoprotein E2 is 90% identical to SEQ ID NO: 30 [SIN-Var1].

49. The composition of embodiment 47 or 48, wherein (i) residue 160 of glycoprotein E2 is absent or is an amino acid other than glutamic acid, (ii) one or more of residues 70, 76 or 159 of the E2 glycoprotein variant is a non-basic residue, and (iii) the E2 glycoprotein variant is not part of a fusion protein with E3 glycoprotein Sindbis virus.

50. The composition of embodiment 49, wherein the glycoprotein E2 is SEQ ID NO: 30 [SIN-Var1].

51. The composition of any one of embodiments 36-50, wherein the Vpx protein comprises an amino acid sequence that is at least 80% identical to SIVmac Vpx [SEQ ID NO: 44].

52. The composition of any one of embodiments 36-50, wherein the Vpx protein comprises at least 90% amino acid sequence identical to SIVmac Vpx (SEQ ID NO: 44), SIVsm Vpx (SEQ ID NO: 45), SIVrcm Vpx (SEQ ID NO: 46), or HIV-2 Vpx (SEQ ID NO: 47).

53. The composition of any one of embodiments 36-49, wherein the Vpx protein comprises at least 90% amino acid sequence identical to SIVdeb Vpr (SEQ ID NO: 48) or SIVmus Vpr (SEQ ID NO: 49).

54. The composition of any one of embodiments 36-53, wherein the antigen is a tumor-specific antigen or a virus-specific antigen.

55. The composition of embodiment 54, wherein the tumor specific antigen is selected from the group consisting of NY-ESO-1, MAGE, MART-1 / Melan-A, BAGE, RAGE, gp100, gp75, mda-7. , tyrosinase, tyrosinase-related protein, renal cell carcinoma antigen, 5T4, SM22-alpha, carbonic anhydrase I, carbonic anhydrase IX (also known as G250), HIF-1alpha, HIF-2alpha, VEGF, specific membrane antigen of the prostate (PSMA), prostate specific antigen (PSA), prostatic acid phosphates, prostate epithelial antigen with six transmembrane domains (STEAP), NKX3.1, telomerase enzyme, survivin, mesothelin, mutated ras, rearrangement bcr / abl, Her2 / neu, mutated p53, wild type p53, cytochrome P450 1B1, N-acetylglucosaminyltransferase-V, human papilloma virus E6 protein, human papillomavirus E7 protein, carcinoembryonic antigen and alpha-fetoprotein.

56. The composition of embodiment 54, wherein the virus-specific antigen is an HIV antigen, an SIV antigen, an adenovirus antigen, an enterovirus antigen, a coronavirus antigen, a calicivirus antigen, an antigen of distemper virus, an Ebola virus antigen, a flavivirus antigen, a hepatitis virus antigen, a herpesvirus antigen, an infectious peritonitis virus antigen, an influenzavirus antigen, a leukemia virus antigen , a Marburg virus antigen, an orthomyxovirus antigen, a papillomavirus antigen, a parainfluenza virus antigen, a paramyxovirus antigen, a parvovirus antigen, a pestivirus antigen, a picornavirus antigen, a poliovirus antigen, a smallpox virus antigen, a rabies virus antigen, a reovirus antigen, a retrovirus antigen or a rotavirus antigen.

57. The composition of any one of embodiments 36-56, wherein the genome of the lentiviral vector further comprises a nucleotide sequence encoding a second antigen.

58. The composition of embodiment 57 wherein the first and second antigen are expressed as a fusion protein comprising a peptide A2 of self-cleavage between the two antigens.

59. The composition of embodiment 58, wherein the self-cleaving peptide A2 comprises the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 57.

60. The composition of any one of embodiments 57-59, wherein the first antigen is NY-ESO-1 and the second antigen is MAGE-A3.

61. The composition of embodiment 57, wherein the first and second antigens are expressed from a bidirectional promoter.

Examples

The following examples are provided by way of illustration and are not intended to limit the scope of the description.

Example 1

Particles of lentiviral vectors pseudotyped with Sindbis virus glycoproteins produced in the presence of kifunensin effectively infect cells expressing DC-SIGN

The aim of the following experiments was to try to produce and characterize lentiviral vectors pseudotyped with highly-mannosylated envelope glycoproteins. In doing so, the inventors unexpectedly discovered that kifunensine was much more effective in the production of pseudotyped lentiviral vectors capable of effectively infecting cells expressing DC-SIGN (eg, dendritic cells) using significantly smaller concentrations in comparison with other mannosidase I inhibitors, including DMNJ.

293T cells were transfected with four separate plasmids encoding the lentiviral genome, Gag / Pol, Rev and the envelope, respectively, using polyethylenimine (PEI). Five hours after the transfection, it was separated the mixture + medium. The medium was added back to the container together with the indicated amount of mannosidase inhibitor (ie, DMNJ, kifunensin and swainsonin). 48 hours later, the supernatant liquid (containing the vector) was collected and filtered with a 0.45 μm filter. HT1080 cells stably expressing the human DC-SIGN receptor were then transduced with the indicated vector volumes. The original HT1080 cells (lacking DC-SIGN) were used as controls and were not transduced by any of the vectors. 48 hours after transduction, the expression of GFP (gfp%) was analyzed in the cells. The results are shown in Figures 1A (HT1080 expressing DC-SIGN) and 1B (HT1080 original).

Particles of lentiviral vectors pseudotyped with Sindbis virus glycoproteins and produced in the presence of low concentrations of kifunensine (1 pg / ml) unexpectedly transfected cells that express DC-SIGN significantly better than those produced in the presence of higher concentrations of DMNj (400 pg / ml) or swainsonin (10 pg / ml). Accordingly, the production of lentiviral vector particles pseudotyped with Sindbis virus glycoproteins in the presence of the mannosidase inhibitor I kifunensin results in significantly greater infection of cells expressing DC-SIGN compared to particles produced in the presence of other inhibitors of the mannosidase I.

Example 2

Low amounts of kifunensine are required to generate particles of pseudotyped lentiviral vectors that effectively infect cells expressing DC-SIGN

The objective of this experiment was to determine the most effective concentration of kifunensine to produce pseudotyped lentiviral vector particles capable of infecting cells expressing DC-SIGN.

293T cells were transfected with the plasmids described in Example 1 using PEI. Five hours after transfection, the mixture + medium was separated. The medium was added back to the container together with the indicated amount of kifunensine (pg / ml), or with 400 pg / ml of DMNJ. 48 hours later, the supernatant liquid (containing particles of lentiviral vectors) was collected and filtered with a 0.45 μm filter. HT1080 cells stably expressing the human DC-SIGN receptor were then transduced with the indicated volumes of supernatant liquid containing the vector. The original HT1080 cells, which were not transduced by the vector, were used as controls. 48 hours after transduction, the expression of gfp (gfp%) was analyzed in the cells. The results are shown in Figures 2A (HT1080 cells expressing DC-SIGN) and 2B (original HT1080 cells).

The particles produced in the presence of 0.125 pg / ml kifunensine matched the capacity of the particles produced in the presence of 400 pg / ml DMNJ to infect cells expressing DC-SIGN (FIG. 2A). The particles produced in the presence of all kifunensine concentrations exceeding 0.125 pg / ml infected cells expressing DC-SIGN much more efficiently than the particles produced in the presence of 400 ng / ml DMNJ. The kifunensine titration showed that the capacity of the particles of lentiviral vectors pseudotyped with Sindbis virus glycoproteins to infect cells expressing DC-SIGN reaches the maximum with particles produced in the presence of 0.25 pg / ml.

Example 3

The objective of this experiment was to characterize the glycosylation profile of pseudotyped lentiviral particles produced in the presence of DMNJ or kifunensine.

The particles of lentiviral vectors pseudotyped with Sindbis virus glycoproteins were prepared according to example 1 in the presence of 1 pg / ml of kifunensine, DMNJ, or without mannosidase I inhibitor. The particles were incubated with PNGasaF or EndoH for 1 hour . PNGaseF is a general endoglycosidase that will cleave all N-linked glycosylation independently of the glycosylation profile (see Figure 3A). EndoH is a specialized endoglycosidase that will only cleave N-linked glycosylation with high mannose content (see Figure 3A). When viral particles are produced in the presence of a mannosidase I inhibitor, the viral envelope is expected to have glycoproteins with a high Mang content and susceptible to being cleaved by EndoH. The samples were analyzed using a gel shift assay performed on an SDS-PAGE gel and immunoblotted with antibody against the viral envelope of Sindbis. The results are illustrated in Figure 3B. The degree of mobility of the viral envelope (SIN-Var1) of the virus produced in the presence or absence of kifunensine or DMNJ, combined with the treatment of PNGasaF or EndoH, is indicative of the degree of glycosylation of Var1. The control virus (band 1) is glycosylated (consequently, it moves more slowly in the gel) and this glycosylation can be completely eliminated by PNGasaF (as evidenced by the more rapid mobility observed in band 2). As expected due to the ability of PNGasaF to cleave any N-linked glycosylation, the virus produced in the presence of JNMD or Kifunensin is sensitive to PNGaseF treatment (lanes 5 and 8). However, only viruses produced in the presence of mannosidase I inhibitors (Var1 DMNJ or kifunensine) are sensitive to treatment with EndoH (lanes 6 and 9) while the control virus (Var1) is only partially sensitive to EndoH. The partial sensitivity probably comes from sites on E2-Var1 that are not normally exposed to mannosidase I during production and do not contribute to the binding to dendritic cells. These results indicate that the efficacy of the mannosidase inhibitor I kifunensin in the The production of viral particles with glycoproteins with a high content of mannose can be measured using a gel displacement test after treatment with EndoH and comparing their efficiency with the particles produced in the presence of DMNj.

Example 4

The content of mannose in the envelope glycoproteins correlates with the concentration of kifunensin in the media used to prepare viral particles

The objective of this experiment was to characterize the glycosylation profile of pseudotyped lentiviral particles produced in the presence of varying concentrations of kifunensine.

The particles of lentiviral vectors pseudotyped with Sindbis virus glycoproteins were prepared according to Example 1 with varying concentrations of kifunensine or 400 pg / ml of DMNJ. The particles were incubated with EndoH for 1 hour. The samples were then analyzed using a gel shift assay and immunoblotting with antibody against the Sindbis virus envelope. In parallel, HT1080 cells stably expressing the human DC-SIGN receptor were transduced with the indicated volumes of pseudotyped lentiviral vector particles prepared with or without kifunensine or DMNJ. 48 hours after transduction, the expression of GFP (shown on the y-axis as a percentage of GFP-positive cells) was analyzed in the cells to create the graphs. The results are illustrated in figure 4.

The degree of mannose content correlates with the degree of transduction of the HT1080 DC-SIGN cells, as indicated by the degree of displacement in the gel of the samples treated with EndoH (figure 4A) and the graphs of the GFP transduction percentage ( figure 4B). That is, increasing concentrations of kifunensine in the media used to prepare viral particles resulted in envelope glycoproteins with higher mannose content, as shown by the greater displacements with EndoH treatment and the higher GFP expression (ie, infection). ) in HT1080 cells expressing DC-SIGN. These results indicate that kifunensin directly affects the degree of mannose content in the viral envelope and this directly correlates with the ability to transduce HT1080 cells expressing the human DC-SIGN receptor. Example 5

Confirmation of the expression of Vpx in particles of pseudotyped lentiviral vectors

The objective of this experiment was to determine if SlVmac Vpx could be expressed and detected in particles of pseudotyped lentiviral vectors.

SlVmac Vpx with an N-terminal HA marker was cloned into a mammalian expression vector driven by a CMV promoter (construct called pENV-SIVmacVpx). To confirm that the Vpx protein was expressed, 293T cells were transfected with this construct and lysed 24 hours after transfection. The lysates were analyzed by immunoblotting using an anti-HA antibody (Figure 5A). To confirm that Vpx was packaged in lentivirus particles, lentiviruses were prepared using four transfected plasmids in 293T packaging cells. These four plasmids encode the lentiviral genome, Gag / Pol (either competent integration [lnt +] or defective integration [Int-]), Rev and the envelope. A fifth plasmid was included for Vpx or not. The virus was harvested two days after transfection and concentrated using centrifugation. 100 ng of p24 was loaded per well into a gel for immunoblotting with anti-HA antibody (Figure 5B). Anti-p24 antibody was used as charge control.

In 293T cells transfected with the plasmid encoding the Vpx gene, the Vpx protein is expressed efficiently (Figure 5A). Similarly, Vpx is packaged into lentivirus particles of both competent integration (Int) and defective integration (Int-) (Figure 5B).

Example 6

Vpx is necessary for the efficient transduction of human dendritic cells by pseudotyped lentiviral vector particles-VSV-G deficient integration

The objective of this experiment was to determine if Vpx was necessary for a productive infection of dendritic cells by particles of pseudotyped lentiviral vectors-VSV-G deficient integration.

Human peripheral blood mononuclear cells (PBMC) were enriched in CD14 + monocytes, followed by enrichment in dendritic cells using GMCSF and IL-4. These human dendritic cells derived from PBMC were transduced with increasing amounts of pseudotyped VSV-G lentiviral particle particle constructs deficient (0.2 ng, 2 ng, 20 ng or 200 ng of p24) containing or not containing Vpx . Five days after infection, the transduction events were measured by regulating cells that were positive for CD11c and evaluating the percentage of cells positive for GFP (x-axis) with DC-SIGN in the y-axis. AZT (a reverse transcriptase inhibitor) was used in the highest dose of lentiviral vector particles (200 ng).

The results are illustrated in Figure 6. Vpx was necessary for poorly integrating pseudotyped VSV-G lentiviral particles to transduce human dendritic cells derived from PBMCs. Efficient transduction depends on reverse transcription because it was inhibited by AZT.

Example 7

Vpx improves the transduction of human dendritic cells by particles of pseudotyped lentiviral vectors-VSV-G of competent integration

The objective of this experiment was to determine if Vpx was necessary for a productive infection of dendritic cells by particles of pseudotyped lentiviral vectors-VSV-G of competent integration.

Human PBMC were enriched in CD14 + monocytes, followed by enrichment in dendritic cells using GMCSF and IL-4. These human dendritic cells derived from PBMC were transduced with increasing amounts of pseudotyped lentiviral particle constructs-VSV-G of competent integration (0.2 ng, 2 ng or 20 ng of p24) containing or not containing Vpx. Five days after infection, the transduction events were measured by regulating the cells that were positive for CD11c and evaluating the percentage of GFP-positive cells (x-axis) with CD11c on the y-axis. Nevirapine (Nev, a reverse transcriptase inhibitor) was used in the highest dose of lentiviral vector particles (20 ng).

The results are illustrated in Figure 7. Vpx improved the ability of competent integration lentiviral vector particles to transduce human dendritic cells derived from PBMC. Improved transduction depends on reverse transcription because it was inhibited by nevirapine.

Example 8

Vpx and the highly mannosylated envelope glycoproteins are necessary for the efficient transduction of human dendritic cells by particles of lentiviral vectors pseudotyped with glycoproteins of the Sindbis virus envelope

The objective of this experiment was to test the capacity of particles of lentiviral vectors pseudotyped with E2 glycoproteins of Sindbis virus that comprise a Vpx protein and produced in the presence of kifunensine, to productively infect dendritic cells.

Human PBMC were enriched in CD14 + monocytes, followed by enrichment in dendritic cells using GMCSF and IL-4. These human dendritic cells derived from PBMC were transduced with varying amounts of constructs of defective integration lentiviral vector particles pseudotyped with SINvar1 (0.2 ng, 2 ng or 20 ng of p24) that either did not contain Vpx, or were produced in the presence or absence of the mannosidase inhibitor I, kifunensine. Five days after infection, the transduction events were measured by regulating the cells that were positive for CD11c and evaluating the percentage of cells positive for GFP (x-axis) with DC-SIGN or CD11c on the y-axis. Nevirapine (Nev, a reverse transcriptase inhibitor) was used in the highest dose of lentiviral vector particles (20 ng).

As shown in Figure 8, unexpectedly, both Vpx and the production of viral particles in the presence of kifunensine are required for efficient transduction of human dendritic cells using a lentivirus pseudotyped with Sindbis virus glycoproteins. Accordingly, these results show that the particles comprising the combination of highly-mannosylated glycoproteins (a result of particle formation in the presence of kifunensine) and Vpx act synergistically to efficiently infect and express the proteins encoded by the lentiviral genome. That is, if one lacks Vpx or highly-mannosylated glycoproteins from the defective integration lentiviral particles pseudotyped with Sindbis envelope glycoproteins, the dendritic cells do not transduce efficiently.

Example 9

Quantification of mannosylation of the envelopes of lentiviral vector particles

The particles of lentiviral vectors pseudotyped with Sindbis virus glycoproteins were prepared according to example 1 without treatment (figure 9A, bands 1-3), 400 pg / ml of DMNJ (figure 9A, bands 4-6), or 1 pg / ml of kifunensine (figure 9A, bands 7-9). The particles were not treated (lanes 1, 4, 7), incubated with EndoH for 1 hour (lanes 2, 5, 8) or incubated with PNGasaF for 1 hour (lanes 3, 6, 9). The samples were then analyzed using a gel shift assay and immunoblotting with antibody against the Sindbis virus envelope.

The analysis of the gel shift data was performed using the Quantity One software from Biorad. In summary, using the "Band" function, vertical lines were drawn along each of the 9 bands shown in Figure 9A. This specifies the program the area to be analyzed. Then, using the "Band Attributes" function, the maximum intensity for each band was determined. These values are also shown in the Figure 9A. This gives us the maximum intensity value of each band in the gel along the "band" indicated previously.

Next, the graphs were assembled based on the intensity profile of each band and its location in the band. The results are shown in Figure 9B (no treatment with mannosidase), Figure 9C (treatment with JNDM) and Figure 9D (treatment with kifunensine).

Each graph represents each of the envelope types digested without enzyme, Endo H or PNGasaF, as indicated. The Y axis is the intensity of the band and the X axis is the location of the band along the described band (ie the value of the "relative front" (rf)), the value of rf is the distance of the band from the top of the gel with respect to the total length of the band).

To quantify the displacement, the maximum intensity of a band that was not cut with a digestive enzyme (ie, complete glycosylation) was compared to a band that was cut with the enzyme PNGasaF (ie, all glycosylation sites were removed). . Therefore, a maximum intensity that is equal to a band that has not been digested with any enzyme will be a displacement of 0% and a maximum intensity that is equal to a band that has been digested with PNGasaF will be considered a 100% displacement . Without the addition of kifunensine (band 2), the EndoH gel shift is 36% of a displacement of PNGaseF. Significantly, the sample treated with kifunensine digested with EndoH (ie, specific digestion of high mannose content) was displaced 90% of the displacement distance of PNGasaF. Accordingly, almost all the glycosylated sites in the envelope of the viral vector are in a high mannose state after treatment with 1 pg / ml of kifunensine.

All the quantitative analysis is shown in Table 1.

Table 1

The spectrum intensity value is the maximum intensity value minus the maximum intensity value of the digested envelope without enzyme. This is done to normalize the values in a 0% displacement spectrum to 100% displacement, as explained above. The% displacement is the value of the intensity of the spectrum divided by the value of the intensity of the spectrum of the samples treated with PNGasaF multiplied by 100. This generates a percentage that quantifies the displacement of a band cut with EndoH in relation to the bands treated without enzyme or with PNGasaF. The envelopes treated with DMNJ were excluded from this analysis because they had heterogeneous N-linked glycosylation patterns.

Example 10

Viral vectors with poorly integrated design elements

For clinical applications that require direct administration of viral vectors but do not require sustained expression of the gene delivered by the vector, such as for vaccines and antigen-directed immunotherapies, deficient integration lentiviral vectors represent an adequate and viable alternative to lentiviral vectors of competent integration for the supply of its genetic load. The impact rate of the D64V integrase mutation within the gag / pol gene and the elimination of cPPT within the vector genome alone and in combination, on the rate of integration of a viral vector were tested.

Materials and methods

Quantification of integration by Alu-PCR.

HuDC-SIGN 293T cells seeded with 5E5 cells / well in 6-well plates were transduced in triplicate with 2E9 genomes per vector well. At 48 hours after transduction, the cells were harvested and the genomic DNA was extracted using the DNeasy kit (Qiagen, Valencia, CA). The genomic DNA was analyzed using a nested PCR assay based on Alu-LTR, which amplifies only the provirus sequences that have been integrated into the genomic DNA of the host. The following modifications were introduced in the previously published method from Brussel et al., Methods Mol. Biol. 304, 139 (2005). Platinum Taq (Life Technologies, Grand Island, NY) was used for the first batch of amplification in a final reaction volume of 25 pl. The cycle conditions of the PCR of the first batch were the following: a denaturation step of 2 min at 95 ° C and then 20 cycles of amplification (95 ° C for 30 s, 55 ° C for 30 seconds, 72 ° C for 90 seconds). Nested PCR was performed using EXPRESS qPCR Supermix Universal (Life Technologies) and 100 nM MH60310 probe in a final volume of 25 pl. The nested PCR protocol began with a 2 minute maintenance at 50 ° C and a 10 minute denaturation step at 95 ° C, followed by 40 cycles of amplification (95 ° C for 15 seconds, 60 ° C for 30 seconds) . All amplification reactions were carried out using the Bio-Rad CFX (model -96 or -384, Bio-Rad Laboratories, Hercules, CA). The copy number of the integrated provirus was calculated with respect to a standard curve generated by parallel nested Alu-PCR of a reference 293T cell line containing integrated proviruses of a known number of copies, diluted in a range of 5-log. The total genomic DNA in the standard curve was normalized by mixing with the genomic DNA of untransduced cells; each standard and unknown sample contained 100 ng of total genomic DNA. This assay allowed the detection of 58 proviruses (Experiment 1) or 4 proviruses (Experiment 2) in 100 ng of genomic DNA.

Quantification of integration by resistance to neomycin.

The vectors encoding the GFP-T2A-NeoR antigen were analyzed independently to determine the rate of integration by formation of neomycin-resistant colonies. The huDC-SIGN HT1080 cells were transduced in 6-well plates with 0.5 ml of serial diluted vector (normalized by genomes) for 2 hours, after which 2 ml of complete medium was added. At 24 hours after transduction, the cells were fed medium containing 800 pg / ml G418 (Life Technologies, Grand Island, NY). The cells were then grown without passage for 11-13 days under selection with G418, after which the colonies were visualized by staining with crystal violet (BD Biosciences, Rockville, MD). The total integration events were calculated as follows: (number of colonies) * (dilution factor) = Integration events.

Quantification of integration by GFP expression.

For the vectors encoding the GFP-T2A-NeoR antigen, the relative integration rate was measured by the expression of GFP over time in mass culture. HT1080 huDC-SIGN cells were transduced in 6-well plates with equal amounts of vector WT / 703 or D64V / 704 (normalized by genomes) in 0.5 ml for 2 hours, followed by the addition of 2 ml of complete medium. The transduced cells were maintained in a medium without drug selection for up to 30 days, with passages at regular intervals. During this period, the expression of GFP was analyzed periodically in the cells by flow cytometry (Guava EasyCyte Plus, Millipore, Billerica, MA).

Results

The huDC-SIGN 293T cells were transduced with pseudotyped vectors with VSV-G integrase-D64V or WT that package WT genomes ("703") or with cPPT ("704") elimination. At 48 hours after transduction, the presence of integrated proviruses was analyzed in the cells by a nested Alu-PCR analysis. As shown in Figure 10A, vectors WT / 704 and D64V / 703 had integration rates that decreased by approximately 2 logs compared to vector WT / 703. In comparison, the integration rate of vector D64V / 704 was reduced by more than 2 logs. These results demonstrate that the genome ID-VP02 (particles of lentiviral vectors pseudotyped with glycoprotein E2 of Sindbis virus with SIVmac Vpx and highly-mannosylated envelope glycoproteins and prepared using packaging cells comprising the gag / pol independent rev system described in FIG. example 12) has a significantly reduced potential integration, and that elements D64V and 704 independently contribute to this phenotype.

To complement the nested Alu-PCR analysis, two additional methods were used to investigate the genome integration rate of the viral vector. In both methods, huDC-SIGN HT1080 cells were transduced with the WT / 703 or D64V / 704 vector encoding GFP and neomycin resistance (NeoR) separated by a self-cleaving T2A linker (GFP-T2A-NeoR). Transduction with any of these vectors results in the expression of GFP and NeoR. The integration rate was measured as a function of antigen expression, either by the growth of neomycin-resistant colonies after selection by G418 or by the expression of GFP over time in mass culture.

In the first method of measuring the integration rate (ie resistance to neomycin), HT1080 huDC-SIGN cells were transduced with serial dilutions of the vector and cultured without passage in the presence of selection by G418. The input vector was normalized with respect to the number of copies of the genome. It was assumed that cells expressing NeoR and surviving prolonged exposure to G418, forming colonies, harbored integrated proviruses. These colonies were counted and the total integration events were calculated. Using this experimental procedure, the integration rate of vector D64V / 704 was reduced by 3 logs with respect to that of WT / 703, in two independent experiments (Figure 10B).

In the second method (ie, GFP expression), the transduced cells were subjected to serial passage in the absence of selection and analyzed by flow cytometry at different times after transduction.

On day 2 after transduction, approximately 40 percent of the cells transduced with the vector WT / 703 were positive for GFP (FIG. 10C). This population remained uniform for the duration of the experiment, suggesting that the expression of GFP was mainly from the integrated proviruses. In contrast, the percentage of GFP-positive cells transduced with the vector D64V / 704 was reduced approximately 100 times on day 6 after transduction and remained low, although superior to the control transduced in a simulated manner, during the rest of the experiment. These results suggest that most transduction events D64V / 704 produced non-integrated vector DNA, which expressed GFP in early times after transduction, but was lost during subsequent cell divisions. The small percentage of cells expressing GFP that remained on day 9 after transduction probably represents the minority of transduction events that produced integrated proviruses. Upon completion of the experiment (day 30), it was calculated that the vector D64V / 704 had diminished in 386 times its capacity to undergo integration, in comparison with the vector WT / 703. These findings are comparable to the results of the nested Alu-PCR analysis.

Taken together, the results of the three methods of measuring the integration rate (nested Alu-PCR, growth of NeoR colonies and% expression of GFP) show that the genome integration rate of the viral vector is reduced by 2-3 logs with respect to the lentiviral vector of 3rd generation, of standard competent integration (WT / 703).

Example 11

Pseudotyped lentiviral vector particles specifically transduce dendritic cells in a homogeneous population of cells

The specificity of the viral vector for dendritic cells was evaluated in the context of a heterogeneous population of potential target cells. Human PBMC were cultured in the presence of GM-CSF and IL-4 for three days to generate a pool of primary cells that included a sufficient number of DCs derived from monocytes expressing DC-SIGN. On the third day, 20 ng of p24 pseudotyped lentiviral vector encoding GFP that were produced in the presence of kifunensine and contained Vpx were added to the culture. Three days after the introduction of the vector in the culture, the expression of GFP was analyzed in the cells as a measure of transduction within the main populations of cells present at the time of analysis: DC (CD11cpos) 6%, B cells ( CD11cneg, CD19pos) 10%, and T cells (CD11cneg, CD3gpos) 80%. As shown in Figure 11, 42% of the cells within the CD11chi, DC-SIGN + population were transduced compared to 0.1% for the populations of both B and T cells present in the culture. Transduction was completely abolished in all cell populations when the reverse transcriptase inhibitor nevirapine (RT inhibitor) was included in the culture. These results demonstrate that in a heterogeneous population of human cells of which DC are a minority, the pseudotyped lentiviral vector specifically transduces DCs expressing DC-SIGN.

Example 12

Design of a gag / pol plasmid independent of Rev

Of the four plasmid system typical of the pseudotyped third generation LVs, two of the plasmids contain sequences within their transcripts that have the potential for recombination. In particular, the transfer vector (referred to herein as the LV genome) and the gag / pol plasmid . There are two regions of sequence homology between the transcripts of the LV genome and gag / pol (figure 12). First, the LV genome has a partial gag sequence after the psi packaging signal consisting of 354 base pairs (bp) that are identical to the 5 'end of the gag sequence in the gag / pol plasmid . The recombination events that occur from this sequence overlap and are called psi-gag recombination events. Second, both the LV genome and gag / pol contain the Rev response element (RRE), which consists of 234 bp that form a secondary RNA structure that allows for the Rev dependent nuclear export of RRE-containing transcripts to the cytoplasm. These two homologous sequences were eliminated by eliminating the gag / pol plasmid RRE and codon optimization of the gag / pol open reading frame (ORF), with the exception of a region of reading frame change between gag and pol that is requires for the translation of pol protein products (figure 12). The reading frame shift region forms a secondary RNA structure at the junction of gag and pol that produces a -1 register shift of the ribosome during translation that is essential for translating pol gene products . Watts et al., Nature, 460: 711-716 (2009). For these experiments, the codons of a 282 bp region between base pairs 1228 and 1509 of the gag / pol ORF were not optimized . This region begins at bp 1563 of the wild type HIV-1 pNL4-3 sequence encoding the Lysine 409 of the Gag protein and extends to include the Gag stop codon. In the remaining regions (pb 1-1228 and pb 1510 4307 gag / pol) the codons were optimized based on the human codon table. Nakamura et al., Nucleic Acids Res, 28: 292 (2000). The complete ORI of RI gag / pol was synthesized in Genscript and cloned in place of the ORF consisting of gag / pol WT and the RRE.

It is known that the removal of the RRE eliminates the Rev dependent export of gag / pol transcripts from the nucleus because the secondary structure of gag / pol RNA retains the transcripts in the nucleus. Banchereau and Steinman, Nature, 392 (6673), 245 (1998). Therefore, codon optimization serves both to eliminate these secondary retention structures and to minimize sequence homology with the partial gag in the LV genome.

Due to the fact that these modifications hypothetically relieve the gag / pol transcript of the need for Rev, the scheme is called Rev independent gag / pol (RI gag / pol), although Rev is still necessary during the production of the vector.

Example 13

The nuclear export of RI gag / pol does not require Rev

To demonstrate that the RI gag / pol transcript is indeed independent of Rev, 293T cells were transfected with wild type gag / pol plasmids (WT gag / pol) or RI, in the presence or absence of a plasmid encoding Rev. Materials and methods

Expression of the Gag protein.

293T cells were seeded in a 6-well plate with 1x106 cells / well. Twenty-four hours later, cells were transfected with 0.5 μg of WT gag / pol or RI gag / pol plasmids in the presence of 0.5 μg of Rev plasmid or 0.5 μg of empty main strand plasmid using Lipofectamine 2000 (Invitrogen). Twenty-four hours later, the cells were lysed with cell extraction buffer (Invitrogen, catalog number FNN0011) and analyzed by SDS-PAGE using pre-molded Bis-Tris 4-12% NuPAGE gels (Invitrogen, catalog number NP0321PK2 ) followed by transfer to a nitrocellulose membrane. The blots were then hybridized with anti-p24 antibody (Abcam, catalog no. Ab9071) or anti-actin antibody (Santa Cruz Biotech, catalog no. Sc-130656). Results

The expression of gag protein products was analyzed in cell lysates using SDS-PAGE and Western blotting with anti-p24 antibody. The plasmid RI gag / pol could express p24 and its precursors were present or not Rev, while the transcript WT gag / pol required Rev for the expression of proteins (figure 13). The processing of the p55 Gag protein appeared different between the RI and WT gag / pol transcripts as a function of the proportion of protein p55: p24, which suggests effects of codon optimization on the expression and / or processing of proteins. These results indicate that transcripts can undergo nuclear export in the absence of Rev, confirming that design changes in the RI gag / pol construction alleviate the requirement for Rev.

Example 14

RI gag / pol produces infectious vector with titles comparable to WT gag / pol

Previous studies have described reductions in the titers of the vectors produced in the absence of Rev. See Gasmi et al., J.Virol. 73: 1828-1834 (1999); Lucke et al., J.Virol. 79: 9359-9362 (2005). The vector made with the gag / pol construction "independent of rev" was tested to determine if it would generate infectious particles with titers comparable to those of WT gag / pol .

Materials and methods

Production of vectors

The vector was produced on a large scale (CF10) or on a small scale (10 cm plate). For large-scale production, 293T cells were seeded in 5E8 cells / 1 liter in a 10-cell Cell Factory device (Nunc, catalog no 140400) in DMEM medium containing 5% serum, L-glutamine and antibiotics . Three days later, the cells were transfected using PEI (stock solution 1 mg / ml) and total plasmid DNA in a ratio of 3: 1 (ml of PEI: mg of DNA). For each 10-cell Cell Factory, 1 mg of the vector's genomic plasmid and 0.5 mg of the remaining plasmids (gag / pol, Rev and VSV-G) were used. Five hours later, the media was replaced with 1 liter of serum-free media (Transfx-293 medium, Hyclone catalog No. SH30860.LS). The vector was collected 2 and 3 days after transfection. The collections were clarified using a prefilter and a 0.45 jm Stericup filter (Millipore). The vector was concentrated by centrifugation in a 1 liter centrifuge bottle at 16,000 g for 5 hours. The sediment of each liter of harvest was resuspended in 1 ml of HBSS and aliquoted for storage at -80 ° C, or resuspended in 1 ml of buffer for treatment with benzonane (Tris-HCl 50 mM at pH 7.5, 1 mM MgCl 2, 5% sucrose v / v). The nuclease benzonane was added in a final concentration of 250 U / ml and incubated overnight at 4 ° C in order to degrade any plasmid left over from the transfection. The benzonane-treated vector preparations were re-concentrated using a pad of sucrose (30% sucrose above, 5% sucrose below) and centrifuged at 116,000g in an ultracentrifuge for 1.5 hours at 4 ° C. The vector pellet was resuspended in 1 ml of HBSS, aliquoted and stored at -80 ° C. For the production of small scale vectors, 293T cells were seeded in a 10 cm dish at 2.5E5 cells / dish and transfected the next day using PEI in a similar manner as described above, but with 6 jg of plasmid of vector genome and 3 jg of remaining plasmids, except in variable amounts of gag / pol plasmids when the vector production is compared. Small-scale transfections were performed in triplicate for greater precision. Five hours later, they replaced the media with 4 ml of DMEM medium containing 5% serum, L-glutamine and antibiotics. The vector was collected 2 and 3 days after transfection and rinsed using a 0.45 μm filter. The vector was stored at -80 ° C.

Quantification of vectors - p24 assay

Quantitation of p24 was carried out using the HIV-1 p24 ELISA kit from Advanced Bioscience Laboratories (Rockville, MD), following the manufacturer's instructions.

Quantification of vectors - GFU assay

293T cells with 2E5 cells / well were plated in a 12-well plate in 1 ml of DMEM medium containing 5% serum, L-glutamine and antibiotics. Twenty-four hours later, the cells in each well were transduced with 2-fold dilutions of the vector encoding GFP. Each vector quantity is prepared in a final volume of 1 ml in DMEM complete medium. Five serial 2-fold dilutions of the supernatant liquid containing vector starting from 200 μl of vector per well were prepared. As a control to rule out pseudotransduction, the 10 pM nevirapine reverse transcriptase inhibitor with the highest vector volume in a parallel well was used. Forty-eight hours after transduction, the expression of GFP was analyzed in the cells in a Guava machine (Guava technologies, now Millipore). The green fluorescence units (GFU) per ml were calculated using a best fit (least squares) linear regression model based on the vector volumes and resulting GFP percentage values in order to predict the number of positive cells for GFP per ml of vector using the FORECAST function in EXCEL. Events that resulted in less than 1% of GFP-positive cells were established as the limit of quantitation (LOQ).

Results

Two preparations of parallel vectors pseudotyped with VSV-G were generated using a vector genome encoding the green fluorescent protein (GFP) as a marker, and with two amounts of input DNA from constructs of WT gag / pol or RI gag / pol. In both vector preparations, p24 was analyzed and shown to have comparable physical particle titers, independently of the gag / pol plasmid used to produce them (Figure 14A). Then, their ability to transduce target cells was tested in these preparations. When normalized by volume, vector RI gag / pol gave comparable transduction events to WT gag / pol for both gag / pol (3 pg or 6 pg) plasmid input quantities used during the production of the vector (Figure 14B). These results indicate that the design elements introduced to generate the gag / pol construction independent of rev did not reduce the performance of physical particles or the infectivity of the lentiviral vector.

Example 15

The RI and WT gag / pol vectors generate equivalent immune responses

LVs are commonly used to deliver nucleic acids that encode proteins to different cell types for research applications and in the clinical setting. However, LV directly injectable are also being developed for both gene therapy and antigen-directed immunotherapy. To test whether an RI gag / pol vector would serve as an LV suitable for immunotherapy applications, the immune responses generated against an antigen transgene encoded by the RI gag / pol vector were evaluated.

Materials and methods

Quantification vector - TU test

The transduction units (TU) were determined using an assay in which the transduction events in a target cell line are measured using a quantitative PCR assay that amplifies reverse transcription vector RNA sequences. Serial dilutions of the test samples and reference material were incubated in duplicate in 96-well tissue culture plates in the presence of target 293T cells. The transduction step was carried out both in the presence and in the absence of the reverse transcriptase inhibitor nevirapine as a means to evaluate the background signal that the residual plasmid DNA can provide. One day after transduction, transduced cells were lysed simulated or with vector by the addition of a buffer containing sodium deoxycholate, Tween-20, sodium dodecylsulfate (SDS) and proteinase K. The cell lysates were then sequentially incubated at 37 ° C, 55 ° C and 95 ° C to ensure proteolysis and DNA denaturation. The denatured cell lysates were then analyzed by qPCR using a primer / probe set that was designed to amplify an approximately 400 bp vector genome sequence located in the 5 'direction of the antigen promoter (EXPRESS qPCR Supermix Universal, Life technologies) . The infectivity titer was calculated with reference to a standard curve comprising linearized plasmid DNA containing the target sequences diluted in a range of 7 log (5.3 copies - 5.3 * 106 copies).

Immunizations

C57BL / 6 mice were immunized subcutaneously at the base of the tail on day 0 with 2 * 107, 1 * 108, or 5 * 108TU of LV encoding LV1b, a polyepitope construct containing the restricted epitope H-2Kb of OVA257 (SIINFEKL) (SEQ ID NO: 24), or HBSS vehicle. The aliquots of LV stored at -80 ° C were thawed at room temperature and then kept on ice. The vector was serially diluted in cold sterile HBSS and transported to the animal facility for injection. The mice were placed in a conventional slotted retention device with the base of the tail accessible. The vector was administered by injection of 50 μl with a 0.3 ml insulin syringe of caliber 29 (Becton Dickenson [BD]) inserted subcutaneously on the right side of the base of the tail, approximately 1 cm caudal to the anus. , which produces a smaller but noticeable distention of the skin around the base of the tail.

Intracellular staining of cytokines (ICS)

Spleens were homogenized by pressing through a 70 pM nylon filter. The red blood cells were lysed by hypotonic shock by brief exposure to ice-cooled ultrafiltered water, followed by immediate isotonic restoration with 10x PBS. For cytokine analysis, the cells were stimulated in 96 wells with peptides at a concentration of 1 pg / ml per peptide in complete RPMI (10% FCS, 10 mM HEPES, 2 pM p-mercaptoethanol and L-glutamine) for 5 hours at 37 ° C, 5% CO ² . The peptide OVA257 (SIINFEKL) was manufactured with a purity of 95% by AnaSpec (Fremont, CA). After stimulation, surface staining was carried out in FACS buffer (PBS, 1% FCS, 2 mM EDTA, 0.01% sodium azide) in the presence of blocking antibody FcR 2.4G2 and LIVE / DEAD ® Fixable Near-IR (L / D NIR, Invitrogen). Antibodies used for surface staining in in vivo experiments included anti-CD4 antibody-PerCP-Cy5.5 from mouse (eBioscience) or CD4-Alexa Fluor 700 (eBioscience), CD8-Pacific Blue (eBioscience) and B220-V500 (BD) After surface staining, the cells were fixed with Cytofix® (BD) and stored at 4 ° C overnight in FACS buffer. The cells were then permeabilized with Perm / Wash ™ buffer (BD) containing 5% rat serum (Sigma Aldrich). Antibodies for intracellular staining were diluted with Perm / Wash ™ buffer containing 5% rat serum and added to permeabilized cells. Antibodies included mouse anti-TNF-α antibody-FITC (eBioscience), IFN- ^and -PE (eBioscience) and IL-2-APC (eBioscience). The cells were washed twice with Perm / Wash ™ buffer and resuspended in FACS buffer and analyzed in a 3 laser LSRFortessa device with high performance sampler (BD). The data was analyzed using FlowJo software (TreeStar, Ashland, OR). The viable CD8 T cells were regulated as follows: lymphocytes (FSCint, SSClD), individual cells (SSC-A = SSC-H), live cells (L / D NIRlD), B220 'CD4' CD8 +. Cytokine regulations were based on the 99.9th percentile (0.1% of positive events in unstimulated cells).

Results

Mice were immunized with a dose range of WT or RI gag / pol vectors , containing a wild-type (INT (+)) or mutant D64V (INT (-)) integrase, which encoded a polyepitope construct called LV1b containing OVA ²⁵⁷ (SIINFEKL) (SEQ ID NO: 24) epitope of CD8 T cells restricted to H-2Kb. 12 days after the immunization, OVA ^257- specific CD8 T cell responses were measured in the spleen by ICS for IFN- ^y (FIG. 15). Both for the vectors of competent integration and those of poor integration, vectors RI gag / pol and WT gag / pol generated comparable CD8 T cell responses, confirming that codon optimization of the gag / pol gene had no negative impact in the function of LV as a vehicle for immunotherapy.

Example 16

The vectors RI and WT gag / pol induce protective antiviral immunity

Although it was observed that the primary responses of the CD8 T cells induced by LV were equivalent with RI gag / pol vectors , the following experiments were carried out to determine if the functional immunity induced by these vectors was also similar. To address this, a recombinant live vaccinia virus stimulation was used as a model of viral infection.

Materials and methods

Stimulation with vaccination virus

C57BL / 6 mice were immunized subcutaneously at the base of the tail on day 0 with 5 * 108 TU of the vector encoding LV1b or HBSS vehicle. Four weeks later, mice were challenged intraperitoneally with 1 x 107 TCID ⁵⁰ recombinant vaccinia virus expressing OVA (rVV-OVA), 1E7 TCID ⁵⁰ wild type vaccinia virus (VV-WT) or HBSS vehicle. Five days after the stimulation, the ovaries were collected for the quantification of the viral load by means of a TCID ⁵⁰ assay.

Results

Mice were vaccinated with deficient integrating LV vectors (INT (-)) encoding OVA ²⁵⁷ with WT or RI gag / pol, and then stimulated 4 weeks later with recombinant vaccinia virus encoding OVA (rVV-OVA) or virus Natural type vaccinia (VV-WT) as control (figure 16). Mice vaccinated with RI vectors gag / pol and WT gag / pol showed marked reductions in viral load after stimulation with rVV-OVA. Confirming that the protection was antigen-specific, the viral load after stimulation with VV-WT was similar between the vehicle and the groups treated with LV. These data indicate that LV RI gag / pol can induce memory CD8 T cells that respond to viral stimulation and provide functional immunity.

Example 17

Modifications of RI gag / pol eliminate the psi-gag recombination, but not other recombination events between the vector genome and the gag / pol transcripts.

The RI gag / pol was designed to try to eliminate the psi-gag recombination, thus also minimizing the possibilities of RCL formation for the third generation LV. A nested PCR-based method was used to analyze the genomic DNA of transduced cells with vector integration in order to detect psi-gag recombination.

Methods and materials

Quantification of vectors - genome assay

Genomic RNA was isolated from vector particles using the QIAamp Viral RNA Mini kit (Qiagen, Valencia, CA). To remove contaminating DNA, the extracted nucleic acid was then digested with DNAsaI (Invitrogen) following the manufacturer's instructions. Two dilutions of each RNA sample treated with DNAsaI were then analyzed by quantitative RT-PCR using the quantitative RT-PCR system Ultrasense One-Step RNA (Invitrogen) and the primers and probe-specific vectors previously described. The number of RNA genome copies was calculated with respect to a standard curve composed of linearized plasmid DNA containing the target sequences, diluted in a range of 7 log (10 copies - 1.0 * 107 copies). The genome titer as expressed herein reflects the number of particles of physical vectors, calculated as a function of genomes, with the prediction that each vector particle contains two single-stranded copies of genomic RNA.

Recombination assay of psi-gag

293T cells were plated with 2E6 in a 10 cm dish. The next day, the cells were transduced with 1E11 genomes from the concentrated pseudotyped VSV-G vector made with WT gag / pol or RI gag / pol. Titers for these vectors were 1.2E13 genomes / ml and 1.5 genomes / ml respectively. Forty-eight hours after transduction, the cells were harvested and the genomic DNA was isolated using a DNA kit for blood and cell culture (Qiagen, catalog no. 13323). 100 ng of genomic DNA was used as template for the first PCR run using high fidelity Platinum taq polymerase (Invitrogen, catalog number 11304-011) and the following cycle parameters: 1 cycle at 94 ° C for 2 minutes; 40 cycles at 94 ° C for 30 seconds, 55 ° C for 30 seconds, 68 ° C for 90 seconds; 1 cycle at 68 ° C for 5 minutes. 1 jl (50 jl) of the first PCR was used as an undiluted template (1: 1) or diluted 1: 100 or diluted 1: 1000 for the nested PCR. The cycle parameters of the nested PCR were identical to those used in the first batch. Primers controls were not included for all reactions. The primers used for the PCRs was 378

(TAAGGCCGAGTCTTATGAGCAGC) (SEQ ID NO: 60), 709

(AGGACTCGGCTTGCTGAAG) (SEQ ID NO: 61), 710

(AGCCTGTCTCTCAGTACAATC) (SEQ ID NO: 62), 835

(TGTCTTATGTCCAGAATGCT) (SEQ ID NO: 63), 863 (GCACGGCAAGAGGCGAGG) (SEQ ID NO: 64), and 864 (GCCGGATGTCCAGGATGCTG) (SEQ ID NO: 65). 25 μl of the total 50 μl of the nested PCR was visualized on a 1% agarose gel made with 1xTAE buffer and ethidium bromide. The bands were extracted and the DNA was purified using a gel extraction kit (Qiagen, catalog number 28704) followed by cloning into a TOPO-TA vector (Invitrogen, catalog number K4500-02) and sequencing (Davis Sequencing , CA) using forward and reverse M13 primers.

Results

Using the nested PCR procedure, a first round of PCR was carried out using a forward primer (709) which binds to the 5 'LV genome of the psi packaging signal and a reverse primer (710) which binds within of the region of displacement of the reading frame of both RI gag / pol and WT gag / pol (figure 12). Then the PCR product of this first batch was diluted and used as a template for a second PCR using a nested direct primer (863) that binds to the LV genome, and a nested reverse primer (835 or 864) that binds to the gag region within WT gag / pol or RI gag / pol , respectively. These two reverse primers were designed to join the same region in both constructions, the only differences being due to codon optimization. The amplicon size of a hypothetical psi-gag recombinant would be 937 bp using the primer pairs 863 and 835, or primer pairs 863 and 864.

Preparations of vectors encoding the LV1b polyepitope were generated with the RI gag / pol or WT gag / pol plasmids , and were used to transduce 293T cells. Forty-eight hours after transduction, the genomic DNA was collected and the PCR was carried out, followed by an analysis on an agarose gel. First, a positive control PCR was performed using primers that bind only within the LV genome (primers 709 and 378). It is predicted that the predicted size of the amplicon of the LV genome with these primers will be 1697 bp. Cell cultures transduced with vector WT gag / pol or RI gag / pol both produced the expected amplicon size, thus confirming that the transduction and integration of proviruses are comparable for both vectors (Figure 17A).

Next, nested PCR was carried out for the recombination of psi-gag as described above. The genomic DNA of the cells transduced with the vector WT gag / pol produced a band in the expected size of 937 bp, consistent with the recombination of psi-gag (left half, Figure 17B). In contrast, genomic DNA from cells transduced with RI gag / pol produced a larger but weaker band at 1329 bp (right half, Figure 17B). To determine the nature of the PCR bands, the 937 bp band of WT gag / pol was extracted , as well as the 1329 bp band of RI gag / pol and cloned into TOPO-TA plasmids. Sequencing revealed that the WT gag / pol band was consistent with a psi-gag recombinant in that the first half of the sequence was aligned with the vector genome and the second half was aligned with the gag / transcript. pol that extended beyond the partial gag sequence (Figure 17C). The weakest 1329 bp band of RI gag / pol encoded a sequence consisting of the first 1253 bp that aligned with the LV genome, but the last 77 bp was aligned with a region of RI gag / pol (figure 17D) . These results indicate that the recombination of psi-gag was detectable in cells transduced with the vector WT gag / pol , but not in cells transduced with the vector RI gag / pol . In addition, these results present proof that recombination, although apparently not dependent on the psi-gag sequences, was still detectable in cells transduced with the RI gag / pol vector.

Example 18

Identification of SIGNR1 homolog of DC-SIGN as mouse receptor for SINvar1

Particles of ID-VP02 vectors were investigated to determine if they could use an endogenous mouse receptor for binding and entry. While humans encode DC-SIGN and a paralog, DC-SIGNR, mice have 8 DC-SIGN homologs (designated SIGNR1 to SIGNR8). Of these, it is predicted that six will be attached to the membrane, namely, SIGNR1, -R3, -R4, -R5, -R7 and -R8. According to functional studies, it is described that SIGNR1, SIGNR3 and SIGNR5 (also known as mouse Dc-SIG) are the closest functional orthologs of DC-human SIGn. Therefore, the ability of SINvar1 to mediate binding and entry by these receptors was tested.

HT1080 cells stably expressing SIGNR1, SIGNR3 or mouse SIGNR5 were generated. These cells were incubated with varying concentrations of vector encoding deficient integration GFP that was pseudotyped with SINvar1, SINvar1 high in mannose modified with kifunensine or with pantropic VSV-G. In Example 8, it was established that an envelope SINvar1 produced in the presence of the mannosidase inhibitor I kifunensine is required for the binding of DC-SIGN and the transduction of human D ^c . In this experiment, therefore, HT1080 cells overexpressing human DC-SIGN as a positive control were used. Of the three mouse DC-SIGN orthologs tested, the pseudotyped vector with SINvar1 transduced only cells expressing mouse SIGNR1 and did so in a kifunensin-dependent manner (Figure 18A-D), as had been observed for the human receptor. The efficiency of the transduction of the kifunensin-modified SINvar1 vector into cells expressing human DC-SIGN and mouse SIGNR1 was remarkably similar (Figure 18A, B), indicating that SIGNR1 is a functional orthologous receptor for vector particles ID-VP02 in the mouse.

Example 19

SIGNR1 is expressed in mouse DC in vivo

In order to further investigate the usefulness of the mouse model for functional studies that reflected the expected mechanism of action in humans, experiments were conducted to determine if SIGNR1 was expressed in mouse dendritic cells.

Materials and methods

Expression of SIGNR1 and 5 in vivo

Single spleen cells or groups of 10 popliteal lymph nodes of 5 mice were stained in FACS buffer (PBS, 1% FCS, 2 mM EDTA, 0.01% sodium azide) in the presence of blocking antibody FcR 2.4G2 and LIVE / DEAD® Fixable Near-IR (L / D NIR, Invitrogen). Antibodies used for surface staining included anti-CD4 antibody-PerCP-Cy5.5 from mouse (eBioscience) or CD4-Alexa Fluor 700 (eBioscience), CD8-Pacific Blue (eBioscience) and B220-V500 (BD). After surface staining, the cells were fixed with Cytofix® (BD) and analyzed in a multiparameter flow cytometer LSRFortessa. The individual live cell events (L / D NIR-, SSH = SSA) were subdivided into B cells (B220 + TCRp-), T cells (TCRP +, B220 ') and DC (B220-TCRp-MHC-II + CD11chi). The regulations for the expression of SIGNR5 and SIGNR1 of each of these subsets are they established using negative control stains that lacked specific antibodies of SIGNR1 and SIGNR5, so that the frequencies of the positive events were <0.00.

Results

Suspensions of individual cells from three individual spleens or three groups of popliteal lymph nodes were analyzed to determine the expression of SIGNR1 and SIGNR5 in T cells, B cells and DC. The expression of SIGNR5 was relatively rare in the steady state, being detected only in a small population of B cells (figure 19). In contrast, while the expression of SIGNR1 was limited in the lymphocytes, approximately 10-12% of the DC MHC-II + CD11chi expressed SIGNR1 (Figure 19). Since SIGNR1 is also a functional receptor for the vector particles ID-VP02, these data led to evaluate whether the vector particles ID-VP02 could be specifically targeted to DCs of mice in vivo.

Example 20

The vector particles ID-VP02 are directed to DC from draining lymph nodes of the mouse in vivo.

In order to determine whether the product of a transgene encoded by particles of lentiviral vectors pseudotyped with E2 glycoprotein of the Sindbis virus could be detected in DC after direct administration, particles encoding GFP or a non-fluorescent negative control protein were injected. the pad of the right leg of BALB / c mice receptors.

Materials and methods

Transduction of ID-VP02-GFP in vivo.

Balb / c mice (n = 15 / group) were injected subcutaneously into the paw pad of 3x1010 genomes of vector particles ID-VP02 encoding GFP, control particles encoding NY-ESO-1, or were left without treating. Four days later, draining popliteal lymph nodes or non-draining cervical lymph nodes from 5 mice were pooled (3 groupings per treatment group) and analyzed in live events (individual L / D (L / D NIR-, SSH = SSA) presence of GFP The phenotype of GFP + cells was determined by co-staining with mouse anti-CD11c-PE-Cy7 antibody (eBioscience), MHC-II-Pacific Blue (eBioscience), SIGNR5-PE (eBioscience) and SIGNR1- APC (eBioscience).

Results

Four days after injecting the particles of ID-VP02 vectors encoding GFP, draining popliteal lymph nodes and non-draining cervical lymph nodes of 5 mice (3 groupings per treatment group) were grouped and the expression of GFP was analyzed. Injection of ID-VP02 vector particles encoding GFP, but not a non-fluorescent control protein, led to the detection of GFP + cells in the draining popliteal lymph node but not in the distal lymph node (FIG. 20A). After further analysis of surface markers, approximately 90% of the transduced cells were identified as DC indicated by the expression of CD11c and MHC-II (Figure 20B), and more than one third of these GFP + DCs were SIGNR1 + (Figure 20C) , supporting a probable function for this receptor in the transduction of live mouse DC.

Example 21

The DNA of vector particles ID-VP02 has a limited biodistribution

Although analysis of lymph node cells was consistent with the specific targeting of mouse DC in vivo, biodistribution studies were conducted to establish whether transduction events could be detected in other tissues, in particular non-lymphoid, and characterize clearance kinetics at positive tissue sites.

Materials and methods

Biodistribution of vectors.

C57BL / 6 mice (n = 3 / group) received 3 x 1010 genomes of ID-VP02 that encoded the LV1b polyepitope construct subcutaneously at the base of the tail. The presence of reverse transcription vector DNA was analyzed by qPCR at 1, 4, 8, 21 or 42 days after injection into the following tissues: injection site (base of the tail), spleen, liver, heart, ovaries , brain and draining lymphatic (inguinal) and non-draining (cervical) lymph node. Tissues were processed in Fastprep Lysing Matrix D tubes using a Fastprep-24 homogenizer (MP Biomedicals, Santa Ana, CA) and the genomic DNA of the homogenates was isolated using the Qiagen DNeasy Blood & Tissue Kit (Qiagen Inc., Valencia, CA ). The eluted DNA (200 ng per sample) was analyzed by qPCR in quadruplicate using EXPRESS qPCR Universal Supermix (Life Technologies, Carlsbad, CA) and a primer / probe set designed to amplify a target sequence of 85 bp into the LV1b cassette. All reactions were performed with the Bio-Rad CFX384 and analyzed using the Bio-Rad CFX Manager software (Bio-Rad Laboratories, Hercules, CA). The number of DNA vector copies was calculated with respect to a standard curve composed of the plasmid DNA containing the target sequences diluted in a range of 7 log (101 copies - 107 copies).

Results

Particles of vector ID-VP02 encoding a polypeptide model antigen construct named LV1b were used to determine the biodistribution of the particles. After injection of the vector, the presence of genomes of reverse transcription vectors (a Dn of the vector) can be measured by qPCR using a set of primers and probe specific for the LV1b cassette. The mice were administered 2.8 * 1010 genomes of pseudotyped-LVlb vector particles in a single subcutaneous injection at the base of the tail. In a temporal evolution between 1 and 42 days after the injection, the DNA of the vector was quantified at the site of the injection (base of the tail), draining lymph nodes (inguinal) and non-draining lymph nodes (cervical), spleen, heart, liver, brain and ovaries). In the early measurement times after administration, the vector DNA was detected exclusively at the site of the injection and in the draining lymph node. The signal of the vector in these tissues decreased over time, with no quantifiable signal at 8 days in the draining lymph node and signal just above the limit of quantification (LOQ, 10 copies) at 42 days at the site of the injection (figure 21). These results indicate that the dispersion of vector particles ID-VP02 outside the injection site is limited to the draining lymph node, where it is assumed that their biological activity would occur, and more than 99% of the vector DNA was eliminated in the term of three weeks.

Example 22

ID-VP02 vector particles induce polyfunctional primary and secondary CD8 T cell responses The following experiments were carried out to measure the capacity of lentiviral vector particles pseudotyped with E2 glycoprotein Sindbis virus to generate an antigen-specific immune response in vivo

Materials and methods

Intracellular staining of cytokines (ICS)

Spleens were homogenized by pressing through a 70 pM nylon filter. Red blood cells were lysed by hypotonic shock by brief exposure to ice-cooled ultrafiltered water, followed by immediate isotonic restoration with 10x PBS. For cytokine analysis, the cells were stimulated in 96 wells with peptides at a concentration of 1 pg / ml per peptide in complete RPMI (10% FCS, 10 mM HEPES, 2 pM p-mercaptoethanol and L-glutamine) for 5 h 37 ° C, 5% CO ² . Peptides, including OVA ²⁵⁷ (SIINFEKL) (SEQ ID NO: 24), LCMV GP ³³ (KAVYNFATM) (SEQ ID NO: 66), AH1 (SPSYVYHQF) (SEQ ID NO: 67) and AH1A5 (SPSYAYHQF) (SEQ ID NO: 25) were manufactured with a purity of 95% by AnaSpec (Fremont, CA). In some experiments, as indicated, mouse anti-CD107a-PerCP-eF710 antibody (eBioscience) was included in the stimulation cocktail to capture CD107a translocated on the surface of degranulation T cells. After stimulation, surface staining was performed in FACS buffer (PBS, 1% FCS, 2 mM EDTA, 0.01% sodium azide) in the presence of blocking antibody FcR 2.4G2 and LIVE / DEAD® Fixable Near-IR (L / D NIR, Invitrogen). Antibodies used for surface staining in in vivo experiments included mouse anti-CD4-PerCP-Cy5.5 antibody (eBioscience) or CD4-Alexa Fluor 700 (eBioscience), CD8-Pacific Blue (eBioscience) and B220- V500 (BD). After surface staining, the cells were fixed with Cytofix® (BD) and stored at 4 ° C overnight in FACS buffer. The cells were then permeabilized with Perm / Wash ™ buffer (BD) containing 5% rat serum (Sigma Aldrich). Antibodies for intracellular staining were diluted with Perm / Wash ™ buffer containing 5% rat serum and added to permeabilized cells. Antibodies included mouse anti-TNF-α antibody-FITC (eBioscience), IFN-y-PE (eBioscience) and IL-2-APC (eBioscience). The cells were washed twice with Perm / Wash ™ buffer and resuspended in FACS buffer and analyzed in a 3-laser LSRFortessa with high performance sampler (BD). The data was analyzed using FlowJo software (TreeStar, Ashland, OR). The viable CD8 T cells were regulated as follows: lymphocytes (FSCint, SSClD), individual cells (SSC-A = SSC-H), live cells (L / D NIRlD), B220 'CD4' CD8 +. Cytokine regulations were based on the 99.9th percentile (0.1% of positive events in unstimulated cells) and the regulation of CD107a was based on the 99th percentile.

Results

To evaluate the immunological activity of the ID-VP02 vector particles in vivo, a vector dose range encoding the full length chicken ovalbumin (ID-VP02-OVA) was administered subcutaneously to C57BL / 6 mice and measured the response of OVA ^257- specific CD8 T cells in the spleen by staining of intracellular cytokines (ICS) (Figure 22A). The frequency of effectors of IFN-Y + between splenic CD8 T cells varies from an average of approximately 15% with a dose of 7.0 * 1010 genomes to approximately 1% with 2.7 * 108 genomes, indicating that ID -VP02 induces CD8 T cell responses in a dose-dependent manner in at least a 2 log dose interval.

To determine whether sensitization with ID-VP02 vector particles induced memory T cells that could be remembered by administering ID-VP02 as a homologous booster, animals were sensitized with a median range dose of 1.0 * 1010 genomes and then reinforced with an equivalent dose 35 days after sensitization. At different times of measurement after sensitization and reinforcement, the response of CD8 T cells by ICS was measured. When analyzing the frequency of CD8 + IFN-Y + T cells, it was found that reinforcement with ID-VP02-OVA induced a specific recall response of OVA257 that was both faster and more than twice as large as the primary response ( Figure 22B). These data indicate that the application of ID-VP02 is not limited to a single administration by vector-specific immunity, which is known to be a problem for other viral vectors, such as adenovirus-based vectors.

In addition to staining for IFN-y, the quality of primary and secondary CD8 T cell responses was analyzed by simultaneous staining of two additional cytokines, TNF-a and IL-2, as well as surface translocation of CD107a, a concept correlated with cytotoxic activity. After both sensitization and reinforcement, the majority of responding CD8 T cells had a multifunctional phenotype, as evidenced by elucidation of CD107a, TNF-a and IL-2 (Figure 22C, D). In particular, 35 days after the boost, essentially 100% of the IFN-Y + cells were CD107a +, a majority also expressed TNF-a, and a substantial fraction of these "triple positive" cells also produced IL-2, which indicates the formation T cell memory with high functional quality.

The KLRG1 and CD127 markers, when measured around the maximum of a virus-specific CD8 T cell response, have been associated with short-lived effector cell (SLEC) and precursor memory cell (MPEC) targets, respectively. As observed during infection with LCMV, when the phenotype of CD8 T cells that bind antigen-specific H-2Kb-OVA257 multimer was analyzed on day 9 after immunization, a cell fraction was polarized to any of the phenotypes KLRG1 + CD127 'SLEC or KLRG1 CD127 + MPEC (Figure 22E). Interestingly, 35 days after the sensitization and booster immunizations, although most of the cells had a CD127 + memory phenotype, they were roughly equally divided between the KLRG1 + and KLRG1 'subsets, and both are described as having greater potential of memory in front of SLEC.

Example 23

Memory CD8 T cells induced by lentiviral vector particles pseudotyped with E2 glycoprotein Sindbis virus expand and present antiviral function

To directly evaluate the function of the memory CD8 T cells induced by SINVarl immunization, an alternative antigen cassette called LV1b was used, which encodes the peptide sequences of both OVA ²⁵⁷ and LCMV GP ³³ minima, two epitopes restricted by H- 2Kb robust.

Materials and methods

Multimer MHC-I and memory phenotype analysis.

Splenocytes prepared as described above were stained with MHC-I pentamers H-2Kb-OVA257 (ProImmune, Oxford, UK) in FACS buffer at room temperature for 10 minutes in the presence of 2-4G2 antibody. The cells were washed once and stained with surface antibodies plus LIR NIR for 20 minutes on ice. Antibodies included CD127-FITC, CD44-PerCP-Cy5.5, K ^l R ^g 1-APC, CD8-Alexa Fluor 700 (all from eBioscience), and B220-V500 (BD). The cells were washed, fixed with Cytofix®, and analyzed as before. Within the population of viable CD8 T cells, CD44hi H-2Kb-OVA257 + pentamer events, regulated based on the 99.9th percentile in non-immunized mice, were analyzed to determine their expression of KLRG1 and CD127.

Stimulation with vaccinia virus

C57BL / 6 mice were immunized subcutaneously at the base of the tail on day 0 with a dose range of ID-VP02 encoding LV1b, a polyepitope construct containing the epitopes restricted by H-2Kb OVA ²⁵⁷ (SIINFEKL) ( SEQ ID NO: 24) and LCMV GP ³³ (KAVYNFATM) (SEQ ID NO: 66), or HBSS vehicle. Five weeks later, mice were challenged intraperitoneally with 1x107 TCID ⁵⁰ recombinant vaccinia viruses expressing OVA (rVV-OVA), 1E7 TCID ⁵⁰ wild type vaccinia virus (VV-WT), or HBSS vehicle. Five days after the stimulation, the spleens were collected for the specific ICS of OVA ²⁵⁷ and GP ³³ as described above, and the ovaries were collected for the quantification of the viral load by the TCID ⁵⁰ assay.

Results

As shown schematically in Figure 23A, 35 days after immunization with ID-VP02-LV1b, mice challenged with recombinant vaccinia virus expressing OVA (rVV-OVA), but not wild type vaccinia virus (VV-WT ), showed a remarkable expansion of OVA ^257- specific CD8 T cells (Figure 23B, C), indicating that these memory cells were remembered in a specific antigen-like manner. In addition, rVV-OVA did not expand GP ^33- specific memory cells (Figure 23B), confirming the specificity requirement of antigen within the same animal. In correspondence with the dose-dependent induction of CD8 T cells responding to infection, there was a clear dose-dependent reduction in the viral load of rVV-OVA in the ovaries of infected mice (FIG. 23D). It is important that the only antigenic sequence shared between the LV1b antigen construct and the rVV-OVA stimulation strain was the MHC class I epitope of the SIINFEKL, indicating that the protection was mediated by CD8 T cells. By confirming that the protection was indeed specific for the antigen, the infection with VV-WT was not affected by the immunization.

Example 24

Immunization by SINVar1 provides both prophylactic and therapeutic anti-tumor efficacy

The CT26 tumor cell line is obtained from a spontaneous colon carcinoma in BALB / c mice. An endogenous epitope that can mediate rejection of implanted CT26 tumors is the AH1 peptide. Although MHC-TCR interaction is relatively weak epitope AH1, the altered peptide ligand AH1A5 can stabilize this interaction, leading to greater cell expansion and T ^c D8 antitumor responses. To generate SINVar1 encoding this epitope, an antigen cassette was generated in which the AH1A5 sequence was inserted into the full-length OVA sequence (OVA-AH1A5), as previously described. Brockstedt et al., Proc. NatlAcadSci. USA 101 (38), 13832 (2004).

Materials and methods

In vivo cytotoxicity assay

BALB / c mice (3 per group) were immunized subcutaneously at the base of the tail with ID-VP02 encoding OVA-AH1A5. Twelve days later, the dye-labeled peptide-pulsed target cells were intravenously transferred through the retroorbital sinus to untreated and untreated control and immunized mice. The target cells were prepared from splenocytes that had not received previous treatment by lysis of the red blood cells by hypotonic shock, then the cells were divided into three populations that were pulsed with 1 pg / ml of peptides AH1 (SPSYVYHQF) (SEQ ID NO: 67), AH1A5 (SPSYAYHQF) (SEQ ID NO: 25), or negative control NY-ESO-181-88 (Rg PESRLL) (SEQ ID NO: 68). The cells were washed and then labeled with CFSE 2 pM (Invitrogen) plus one of three concentrations of Cell Trace Violet (Invitrogen): 2 pM, 0.2 pM or 0.02 pM. The target cells were combined in a 1: 1: 1 ratio and 5 * 10® total cells were transferred to the receptors. The next day, the spleens were collected and the relative recovery of each population was compared between previously untreated and immunized mice to calculate specific death as previously described. Wonderlich et al., Curr. Protoc. Immunol. Chapter 3, Unit (2006).

CT26 tumor stimulation

For prophylaxis experiments, BALB / c mice (10 per group) were immunized subcutaneously at the base of the tail with ID-VP02 encoding OVA-AH1A5, an antigen containing a restricted rejection epitope by MHC-I defined for CT26 tumor cells. Four weeks later, immunized and control mice not subcutaneously treated with 8 * 104 CT26 tumor cells were injected into the right flank. Tumor growth was monitored three times a week and the mice were sacrificed when the tumor area exceeded 100 mm2. Test experiments of ID-VP02 in the therapeutic mode were performed in the same manner, with the exception that immunization with ID-VP02 was delayed up to four days after tumor implantation.

Results

When BALB / c mice were immunized with ID-VP02 encoding OVA-AH1A5, dose-dependent induction of multifunctional AH1A5-specific CD8 T cells was observed, approximately half of which cross-reacted with the AH1 sequence endogenous (figure 24 ^a ). The ability of CD8 T cells induced by ID-VP02 to acquire cytolytic capacity was analyzed directly by cytotoxicity assay in vivo. Three populations of splenocyte target cells were labeled simultaneously with CFSE plus variable concentrations of Cell Trace Violet, then pulsed with AH1, AH1A5 or a negative control peptide. The target cells were mixed in a 1: 1: 1 ratio, then co-transferred to recipient mice immunized 12 days earlier with ID-VP02 or left untreated. After 1 day, the relative recovery of the pulsed targets with AH1 and AH1A5 was reduced in mice immunized with ID-VP02, with specific death rates of more than 90% against AH1A5 and approximately 25% against AH1 (FIG. 24B), indicating that ID-VP02 induces functional cytotoxic CD8 T cells against the immunizing antigen.

As the first assay of antitumor efficacy, mice immunized subcutaneously with ID-VP02-OVA-AH1A5 or vehicle were stimulated 28 days later with CT26 tumor cells implanted in the flank. Although all control mice had a lethal tumor growth (> 100 mm2) on day 21, 70% of mice immunized with ID-VP02 could reject the implanted tumors and these surviving mice were tumor-free for at least 60 days (Fig. 24C). These findings were extended by the application of ID-VP02 as therapy to previously implanted CT26 tumors. In this model, the tumors were allowed to grow for 5 days, then the animals were treated with ID-VP02-OVA-AH1A5 or vehicle control. As in the prophylactic experiments, all control animals succumbed to tumor growth in approximately three weeks (Figure 24D). For him In contrast, all mice treated with ID-VP02 showed effects on tumor progression, ranging from a delay in growth kinetics to absolute rejection. The tumors either did not grow until reaching a palpable size (2/10) or completely regressed (3/10) in the immunized group, leading to 50% of the mice being without until at least day 60. These data show that ID -VP02 can exert cytotoxic antitumor activity both in the prophylactic and therapeutic framework, which supports the evaluation of ID-VP02 as a therapeutic agent for cancer in humans.

Claims (14)

A method for generating a pseudotyped lentiviral vector particle comprising: (a) culturing in a culture medium comprising kifunensin, a virus packaging cell comprising: (1) a lentiviral vector genome comprising a polynucleotide encoding a sequence of expressible interest, (2) a polynucleotide encoding an alphavirus envelope glycoprotein that preferentially binds to dendritic cells expressing DC-SIGN and (3) a polynucleotide that encodes a Vpx protein or a Vpr protein that retains the inhibitory activity of SAMHD1; Y (b) isolating a pseudotyped lentiviral vector particle that preferentially binds dendritic cells expressing DC-SIGN, wherein said lentiviral vector particles are more highly mannosylated compared to a control composition of the same pseudotyped lentiviral vector particles prepared in the absence of a mannosidase inhibitor. 2. The method of claim 1, wherein the lentiviral vector particle is of poor integration. 3. The method of claim 1 or 2, wherein the envelope glycoprotein alphavirus is selected from the group consisting of a Sindbis E2 glycoprotein, an E2 Sindbis glycoprotein that is 90% identical to SEQ ID No: 30 [SIN-Var1] an E2 glycoprotein Sindbis which is 90% identical to SEQ ⁱ D NO: 30 [SIN-Var1] wherein (i) the remainder 160 of the E2 glycoprotein is absent or is an amino acid other acid glutamic, (ii) one or more of residues 70, 76 or 159 of the E2 glycoprotein variant is a non-basic residue, and (iii) the E2 glycoprotein variant is not part of a glycoprotein fusion protein. E3 of the Sindbis virus or an E2 glycoprotein of the Sindbis as set forth in SEQ ID NO: 30 [SIN-Var1]. The method of any one of claims 1-3, wherein the Vpx protein comprises an amino acid sequence that is at least 80% identical to SIVmac Vpx [SEQ ID NO: 44], or wherein the Vpx protein comprises a amino acid sequence at least 90% identical to SIVmac Vpx ^(s E ^q ID NO: 44), SIVsm Vpx (SEQ ID NO: 45), SIVrcm Vpx (SEQ ID NO: 46), or HIV-2 Vpx (SEQ ID NO: 47), or wherein the Vpx protein comprises at least a 90% amino acid sequence identical to SIVdeb Vpr (S ^e Q ID NO: 48) or SIVmus Vpr (S ^e Q ID NO: 49). The method of any one of claims 1-4, wherein the expressible sequence of interest encodes an exogenous antigen, wherein said exogenous antigen is optionally: (a) a tumor-specific antigen or a virus-specific antigen; (b) a tumor-specific antigen selected from the group consisting of NY-ESO-1, MAGE, MART-1 / Melan-A, BAGE, RAGE, gp100, gp75, mda-7, tyrosinase, tyrosinase related protein, renal cell carcinoma antigen, 5T4, SM22-alpha, carbonic anhydrase I, carbonic anhydrase IX (also known as G250), HIF-1alpha, HIF-2alpha, VEGF, prostate specific membrane antigen (PSMA), specific antigen of the prostate (PSA), prostatic acid phosphates, prostate epithelial antigen with six transmembrane domains (STEAP), NKX3.1, telomerase enzyme, survivin, mesothelin, mutated ras, bcr / abl rearrangement, Her2 / neu, p53 mutated , natural type p53, cytochrome P4501B1, N-acetylglucosaminyltransferase-V, human papilloma virus E6 protein, human papillomavirus E7 protein, carcinoembryonic antigen and alpha-fetoprotein; or (c) a virus-specific antigen selected from an HIV antigen, an SIV antigen, an adenovirus antigen, an enterovirus antigen, a coronavirus antigen, a calicivirus antigen, a distemper virus antigen, an antigen from Ebola virus, a flavivirus antigen, a hepatitis virus antigen, a herpesvirus antigen, an infectious peritonitis virus antigen, an influenza virus antigen, a leukemia virus antigen, a Marburg virus antigen , an orthomyxovirus antigen, a papillomavirus antigen, a parainfluenza virus antigen, a paramyxovirus antigen, a parvovirus antigen, a pestivirus antigen, a picornavirus antigen, a poliovirus antigen, a smallpox virus antigen, a rabies virus antigen, a reovirus antigen, a retrovirus antigen or a rotavirus antigen. 6. The method of claim 5, wherein the lentiviral vector further comprises a nucleotide sequence encoding a second antigen. The method of claim 6, wherein the first and second antigen are expressed as a fusion protein comprising a peptide A2 of self-cleavage between the two antigens, or wherein the first and second antigens are expressed from a bidirectional promoter. The method of claim 6 or 7, wherein the first antigen is NY-ESO-1 and the second antigen is MAGE-A3. The method of any one of claims 5-8, wherein the genome of the lentiviral vector further comprises a nucleotide sequence encoding a stimulatory molecule selected from the group consisting of GM-CSF, IL-2, IL-4, IL-6, IL-7, IL-15, IL-21, IL-23, TNF-alpha, B7.1, B7.2, 4-1BB, CD40 ligand and drug-induced CD40. The method of claim 1, wherein the polynucleotide of interest encodes a stimulator molecule selected from the group consisting of GM-CSF, IL-2, IL-4, IL-6, IL-7, IL-15, IL -21, IL-23, TNFalpha, B7.1, B7.2, 4-1BB, CD40 ligand and drug-induced Cd40. The method of any one of claims 1-10, wherein the kifunensine is present in the culture medium at a concentration of about 0.1 pg / ml to about 10 pg / ml, or wherein kifunensine is present in the culture medium in a concentration of about 0.25 pg / ml to about 2 pg / ml. The method of any one of claims 1-11, wherein the virus packaging cell further comprises a polynucleotide comprising the gag and pol genes. The method of any one of claims 1-11, wherein the virus packaging cell further comprises: (i) a polynucleotide comprising the gag and pol genes; Y (ii) a polynucleotide encoding a rev. The method of claim 12 or 13, wherein the polynucleotide comprising the gag and pol genes lacks a functional rev response element (RRE) and / or the pol gene encodes an inactive integrase enzyme.